Methods and formulations for treatment of spinobulbar muscular atrophy

ABSTRACT

Methods and formulations are provided for treating spinobulbar muscular atrophy in a subject in need thereof. By administering a therapeutically effective amount of a selective androgen receptor modulator or a small molecule such as 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole or a derivative, prodrug, or pharmaceutically acceptable salt thereof, one or more symptoms of spinobulbar muscular atrophy can be ameliorated. The effective amount can be effective to prevent or delay loss of body weight, a loss of mobility, and/or a loss of physical strength in the subject; to prevent or delay neurogenic atrophy and/or to prevent a loss of spinal cord motor neurons in the subject; to restore the frequency of type I myofibers to normal levels for a healthy subject; and/or to reverse testicular atrophy in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, co-pending U.S.provisional application entitled “METHODS AND FORMULATIONS FOR TREATMENTOF SPINOBULBAR MUSCULAR ATROPHY” having Ser. No. 62/578,084, filed Oct.27, 2017, the contents of which are incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under award NS053825awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

The present disclosure generally relates to formulations and methods oftreatment for spinobulbar muscular atrophy.

BACKGROUND

Spinal bulbar muscular atrophy (SBMA), or Kennedy's disease, is aprogressive neurodegenerative disease affecting approximately 1 in40,000 men worldwide¹; however, this number is likely underestimated dueto common misdiagnoses (e.g., limb girdle muscular dystrophy andamyotrophic lateral sclerosis [ALS])². Although SBMA is not typicallyfatal, the quality of life is profoundly affected as patients experiencebulbar dysfunction, limb weakness loss of ambulation, and partialandrogen insensitivity that often leads to feminization, testicularatrophy and fertility problems. Neuromuscular symptoms generally firstappear as muscle spasms and weakness in the extremities, mouth, andthroat, which progress to muscle wasting due to loss of motor neurons.There is currently no known cure for SBMA, and treatment is symptomatic,usually entailing physical therapy and rehabilitation. Patients withSBMA frequently become confined to a wheelchair later in life andrequire assistance with common daily tasks, such as eating².

There remains a need for methods and treatments of SBMA that overcomethe aforementioned deficiencies.

SUMMARY

In various aspects described herein, pharmaceutical compositions andmethods are provided for treating and/or alleviating one or moresymptoms associated with spinal bulbar muscular atrophy in a subject inneed thereof. In some aspects, the subject is a mammal, and inparticular a human. In some aspects, the methods and the uses of theformulations are effective to prevent or delay loss of body weight, aloss of mobility, and/or a loss of physical strength in the subject. Insome aspects, the methods and the uses of the formulations are effectiveto prevent or delay neurogenic atrophy and/or to prevent a loss ofspinal cord motor neurons in the subject. In some aspects, the methodsand the uses of the formulations are effective to restore the frequencyof type I myofibers to normal levels for a healthy subject. In someaspects, the methods and the uses of the formulations are effective toreverse testicular atrophy in the subject.

In some aspects, pharmaceutical formulations and methods of use thereofare provided where the pharmaceutical formulation includes atherapeutically effective amount of a small molecule, a derivativethereof, a prodrug thereof, or a salt thereof; wherein the smallmolecule has a structure according to the following formula

wherein each occurrence of R¹, R², R³, and R⁴ is independently ahydrogen, a hydroxyl, a halogen, or a substituted or unsubstituted C₁-C₆alkyl or alkoxy; wherein each occurrence of X¹ and X² is independently Oor S; wherein each occurrence of A¹ is independently none or asubstituted or unsubstituted C₁-C₆ alkyl diradical; and wherein thetherapeutically effective amount is effective to ameliorate one or moresymptoms of spinobulbar muscular atrophy in the subject.

In some aspects, the small molecule is1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazolehaving a structure according to the following formula

In some aspects, pharmaceutical formulations and methods of use thereofare provided where the pharmaceutical formulation includes atherapeutically effective amount of a selective androgen receptormodulator, wherein the therapeutically effective amount is effective toameliorate one or more symptoms of spinobulbar muscular atrophy in thesubject. In some aspects, the selective androgen receptor modulatoralters a co-regulator binding to the activation function-2 (AF2) domainof the androgen receptor. In some aspects, the selective androgenreceptor selectively binds to the binding function-3 (BF3) domain of theandrogen receptor.

Other systems, methods, features, and advantages of pharmaceuticalmethods and compositions will be or become apparent to one with skill inthe art upon examination of the following drawings and detaileddescription. It is intended that all such additional systems, methods,features, and advantages be included within this description, be withinthe scope of the present disclosure, and be protected by theaccompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIGS. 1A-1I demonstrate activation function-2 (AF2) modulation rescuesdegeneration in a fruit fly model of Spinal bulbar muscular atrophy(SBMA). FIG. 1A is a structural depiction of the androgen receptor (AR)ligand-binding domain (LBD) in complex with tolfenamic acid (TA) or1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole(MEPB). The binding function-3 (BF3) domain and the core AF2 domain areindicated in the BF3 unbound structure. The TA and MEPB binding aredepicted by the shaded regions in the central and far right structuresrespectively. The modeled FXXLF or LXXLL motifs are labelled. FIG. 1B isa bar graph of the viability of flies expressing human AR52Q inpan-neuronal tissues in the presence of vehicle (far left, white bar),and 1 mM DHT with varying amounts of TA (black bars) ranging, left toright, from 0 μM to 400 μM TA; n=50 adult flies/treatment group. FIG. 1Cis a bar graph of the viability of flies expressing human AR52Q inpan-neuronal tissues in the presence of vehicle (far left, white bar),and 1 mM DHT with varying amounts of MEPB (black bars) ranging, left toright, from 0 μM to 400 μM MEPB; n=50 adult flies/treatment group. FIG.1D depicts representative traces of the walking pattern of adult fliesexpressing AR52Q, AR52Q-K720A, or AR66Q-E897K in motor neurons for 90sec in a well of a 12-well tissue culture plate. Adult flies were rearedon food containing 1% ethanol+0.1% DMSO, 1 mM DHT+0.1% DMSO, 1 mMDHT+100 μM TA, or 1 mM DHT+100 μM MEPB. FIG. 1E is a bar graphquantifying the mean distance walked by adult SBMA flies, determinedfrom 15 individual tracing patterns per genotype or treatment group.FIG. 1F is a bar graph quantifying the mean velocity of adult SBMAflies, determined from 15 individual tracing patterns per genotype ortreatment group. FIG. 1G is an image of staining and quantification ofneuromuscular junctions of larval SBMA flies. Arrowheads indicatesatellite boutons. FIG. 1H is a bar graph of the mean satellite boutonsquantified for 2-3 muscle segments from 12 dissected larval pelts pertreatment group. FIG. 1I is a bar graph of the mean neuromuscularjunction branches quantified for 2-3 muscle segments from 12 dissectedlarval pelts per treatment group. Data shown in FIGS. 1B-1C wereevaluated by Chi-square analysis; comparisons between the actualpopulation frequencies of each treatment group and the predictedpopulation frequency were based on the DHT+DMSO group. Data shown inFIGS. 1E-1F and FIGS. 1H-1I were evaluated by one-way ANOVA, and Dunnettposthoc analysis was used for pairwise comparisons between eachtreatment group and the DHT+DMSO group. *P≤0.05, **P≤0.01. All errorbars represent s.e.m.

FIGS. 2A-2L depict AR121Q-expressing mice recapitulate SBMA symptoms andpathology. FIG. 2A-2D are graphs of phenotypic measures of SBMAdegeneration of NTG (diamonds) and AR121Q mice (squares), in addition tosham-operated (triangles) and castrated (circles) AR121Q mice. FIG. 2Ais a graph of the mean body weight. FIG. 2B is a graph of the meanrotarod activity. FIG. 2C is a graph of the mean grip strength. FIG. 2Dis a graph of the survival. All data points in FIGS. 2A-2D are for n=5mice per group, ***P<0.001 by two-way ANOVA and Dunnett multiplecomparison test (FIGS. 2A-2C) or Kaplan-Meier estimation (log-rank test)(FIG. 2D). FIGS. 2E-2F are images of representative spinal cord (FIG.2E) and skeletal muscle (FIG. 2F) sections from 7-week-old NTG andAR121Q mice. Sections were stained with hematoxylin and eosin (H&E)stain for assessment of morphology, in addition to AR (N20) andubiquitin antibodies. White arrowheads indicate ubiquitin-positivenuclear inclusions in the spinal cord. Dotted lines trace aroundrepresentative myofibers, demonstrating differences in myofiber size.Arrow indicates atrophied myofibers. Arrowheads indicate myofiberscontaining centralized nuclei. FIG. 2G is images of PolyQ (5TF1-1C2) andubiquitin costaining in spinal cord of NTG and AR121Q mice. FIGS. 2H-2Kare bar graphs quantifying the mean muscle fiber diameter ofgastrocnemius/soleus myofibers (FIG. 2H), mean type I and type IIhindlimb muscle fiber staining (FIG. 2I), and ChAT-positive motorneurons in the anterior horn of the thoracic spinal cord (FIGS. 2J-2K)of NTG and AR121Q mice. n=3 mice per genotype, **P<0.01, *P<0.05 byStudent t-test. FIG. 2L is images of the immunofluorescence withantibodies against NCAM and PSA-NCAM in skeletal muscle tissues of NTGand AR121Q mice. Scale bars represent 50 μm. All error bars represents.e.m.

FIGS. 3A-3I demonstrate MEPB improves phenotypic outcomes and pathologicdegeneration in a pilot preclinical trial in SBMA mice. FIG. 3A is agraph of the mean body weight of AR121Q mice from 4 weeks until 8 weeksof age. FIG. 3B is a graph of the mean rotarod performance of AR121Qmice from 5 weeks until 7 weeks of age. FIG. 3C is a graph of the meangrip strength (measured as grams force) of all four paws of AR121Q mice.Data shown in FIGS. 3A-3C were evaluated by one-way ANOVA with repeatedmeasures followed by Tukey posthoc analysis for all pairwisecomparisons. *P≤0.05 vs NTG. FIG. 3D is a graph of the Kaplan-Meiersurvival estimation of AR121Q mice (log-rank test). FIGS. 3E-3F are bargraphs quantifying footprint/gait analysis of AR121Q mice. Averagestride length (FIG. 3E) and forepaw and hindpaw overlap (FIG. 3F) aredepicted. n=1-3 NTG mice per drug group, n=3-5 AR121Q mice per druggroup. Two-way ANOVA followed by Tukey multiple comparison test.*P≤0.05, **P≤0.01 vs NTG. All error bars represent s.e.m. FIG. 3G is aset of video stills of clasping behavior in representative NTG andAR121Q mice. FIG. 3H is images of representative spinal cord sectionsfrom AR121Q mice treated with vehicle, TA, or MEPB. Sections werestained with AR (N20), ubiquitin, or polyQ (5TF1-1C2) antibodies.Arrowheads indicate the presence of ubiquitin-positive nuclearinclusions. FIG. 3I is images of representative skeletal muscle sections(gastrocnemius/soleus) from AR121Q mice treated with vehicle, TA, orMEPB. Sections were stained with H&E or Gomori trichrome stain formuscle morphology, in addition to ubiquitin antibody. Dotted lines tracearound representative myofibers, demonstrating differences in myofibersize. Arrows indicate atrophied myofibers. Arrowheads indicate myofiberscontaining centralized nuclei. Vehicle is 1% DMSO in corn oiladministered three times per week; TA is 50 mg/kg administered threetimes per week; and MEPB is 50 mg/kg administered three times per week.Scale bars represent 50 μm.

FIGS. 4A-4K demonstrate MEPB improves phenotypic outcomes and improvesquality of life parameters in a large-scale preclinical trial in SBMAmice. FIGS. 4A-4F are graphs for each phenotypic assay (mean body weightin FIGS. 4A-4B, mean rotarod activity in FIGS. 4C-4D, mean grip strengthin FIGS. 3E-4F) are depicted using identical scales to allow comparisonbetween NTG mice (FIGS. 4A, 4C, and 4E) and AR121Q mice (FIGS. 4B, 4D,and 4F). Mice were treated with vehicle (squares; 1% DMSO in corn oil),low-dose MEPB (circles; 50 mg/kg, three times per week), or high-doseMEPB (triangles; 100 mg/kg, three times per week). Arrows indicate lastgraphed data point for each treatment group due to attrition of animalnumbers (<3 mice/group) from loss of >10% body weight or limbparalysis/paresis. *P≤0.05, **P≤0.01. All error bars represent s.e.m.FIGS. 4G-4I are bar graphs depicting side-by-side comparisons ofphenotypic assays (mean body weight (FIG. 4G), mean rotarod activity(FIG. 4H), and mean grip strength (FIG. 4I) in vehicle-, low-dose MEPB-,and high-dose MEPB-treated AR121Q mice for time points in which ≥3 miceper treatment group were assayed. Data shown in FIGS. 4G-4I wereevaluated up to 8 or 9 weeks of age by one-way ANOVA with repeatedmeasures followed by Tukey posthoc analysis for all pairwisecomparisons. *P≤0.05, **P≤0.01. FIG. 4J is a graph of a Kaplan-Meiersurvival estimation in treated (vehicle, low-dose, and high-dose) NTGand AR121Q mice (log-rank test). FIG. 4K is a graph of QOL score ofAR121Q mice from each treatment group from 6 to 8 weeks of age. In allpanels, mice were treated with vehicle (1% DMSO in corn oil), low-doseMEPB (50 mg/kg, three times per week), or high-dose MEPB (100 mg/kg,three times per week). A mixed-effect model was applied with SASsoftware to determine statistical significance. All error bars represents.e.m.

FIGS. 5A-5G demonstrate MEPB reduces degeneration in spinal cord andskeletal muscle of SBMA mice. FIG. 5A is images of representative spinalcord sections from AR121Q mice treated with vehicle (1% DMSO in cornoil) or high-dose MEPB (100 mg/kg, three times per week). Sections werestained with H&E and toluidine blue to assess spinal cord morphology.Sections were also stained with AR (N20), polyQ (5TF1-1C2), andubiquitin antibodies. White arrowheads indicate ubiquitin-positivenuclear inclusions. Scale bars represent 200 μm. FIG. 5B is a bar graphquantifying the mean relative number of cells in the spinal cord ventralhorn containing positive polyQ (white) or ubiquitin (black) staining inAR121Q mice. Quantification was performed from 2-4 fields/mouse, 3mice/treatment group. FIG. 5C is images of representative skeletalmuscle (gastrocnemius/soleus) sections from AR121Q mice treated witheither vehicle or high-dose MEPB. Sections were stained with H&E andGomori trichrome to evaluate morphology, in addition to AR, polyQ, andubiquitin antibodies. Dotted lines trace around representativemyofibers, demonstrating differences in myofiber size. Arrows indicateatrophied myofibers. Arrowhead indicates myofibers containingcentralized nuclei. Scale bars represent 200 μm. FIG. 5D is a bar graphquantifying the mean muscle fiber diameter of gastrocnemius/soleusmyofibers of NTG (white) and AR121Q mice (black). Quantification wasperformed from 2-4 fields/mouse, 3 mice/treatment group. Data shown inFIG. 5B and FIG. 5D were evaluated by two-way ANOVA followed by Dunnettposthoc analysis for pairwise comparisons between each MEPB treatmentgroup and vehicle. *P≤0.05. FIG. 5E is representative images of skeletalmuscle stained with antibodies against NCAM and PSA-NCAM in AR121Q micetreated with vehicle (1% DMSO in corn oil), low-dose MEPB (50 mg/kg,three times per week), or high-dose MEPB (100 mg/kg, three times perweek). Scale bars represent 50 μm. FIG. 5F is a bar graph ofPSA-NCAM/NCAM colocalized regions. Data were evaluated by two-way ANOVAfollowed by Tukey multiple comparison test. ***P≤0.001. FIG. 5G is a bargraph of the area of testis were measured in 3 to 4 sections per mouse,in 2 to 3 mice per group. Data were evaluated by one-way ANOVA andDunnett posthoc analysis for comparison between each AR121Q treatmentgroup and NTG vehicle. *P≤0.05. All error bars represent s.e.m.

FIGS. 6A-6D demonstrate AF2 modulation does not inhibit AR functionalactivity. FIG. 6A is representative immunofluorescence of HEK293T cellstransiently transfected with AR65Q for 48 h in culture media devoid ofsteroid hormones. Cells were treated for 24 h with vehicle, 10 nM DHT,or 10 nM DHT+10 μM bicalutamide (Bic), TA, or MEPB prior to staining(n=3) with AR (D6F11) and DAPI. Scale bars represent 10 μm. FIG. 6B isan image of third instar larvae expressing GFP-AR52Q in motor neuronswere dissected and stained for nuclear membrane and DAPI FIG. 6C is abar graph of the AR transcriptional activity reporter assay. HEK293Tcells were transiently transfected with AR24Q or AR65Q in addition to anARE-luciferase reporter prior to treatment with Bic, TA, or MEPB. Fourindependent biological replicates were performed on different days withthree sample replicates for each treatment group. FIG. 6D is a bar graphof the digital PCR assay to access the impact of MEPB on AR target geneexpression. FIG. 6E is a bar graph of the mammalian two-hybrid assay toassess binding between the AR LBD and the corepressors NCoR or SMRT inthe presence of TA or MEPB. Two independent biological replicates wereperformed on different days with three sample replicates for eachtreatment group. Data shown in FIG. 6C were evaluated by two-way ANOVAfollowed by Dunnett posthoc analysis for pairwise comparisons betweeneach treatment group and the DHT+DMSO group. Data shown in FIG. 6D wereevaluated by ordinary one-way ANOVA followed by Tukey posthoc analysisfor all pairwise comparisons. Data shown in FIG. 6E were evaluated bytwo-way ANOVA followed by Tukey posthoc analysis for all pairwisecomparisons. *P≤0.05, **P≤0.01, ***P≤0.001. All error bars represents.e.m.

FIGS. 7A-7F demonstrate screening of AF2-modulating compounds in aDrosophila model of SBMA. FIG. 7A is a bar graph of mean viability ofSBMA flies (AR52Q) reared on food containing vehicle (EtOH), DHT, or DHTin addition to the AF2 modulators described by Estebanez-Perpina et al.(2007)¹⁹. FIG. 7B is a bar graph of mean viability of SBMA flies rearedon food containing vehicle, DHT, or DHT in addition to the AF2modulators described by Lack et al. (2011)²¹. FIG. 7C is a bar graph ofmean viability of transgenic flies expressing AR52Q (ELAV>UAS-AR52Q)reared on food containing vehicle or DHT and transgenic flies expressingAR52Q-K720A or AR66Q-E897K reared on food containing DHT. FIG. 7D is abar graph of mean viability of SBMA flies (ELAV>UAS-AR52Q) reared onfood containing MEPB in the absence of DHT. FIG. 7E is a bar graph ofmean viability of SBMA flies (ELAV>UAS-AR52Q) reared on food containingDHT and bicalutamide. FIG. 7F is a bar graph of mean viability of SBMAflies (ELAV>UAS-AR52Q) reared on food containing DHT and ibuprofen. Alldata were evaluated by Chi-square analysis; n=50 adult flies/treatmentgroup for all experiments. Comparisons of data shown in FIG. 7A, FIG.7B, FIG. 7C, and FIG. 7E were made between the actual populationfrequencies of each treatment group and the predicted populationfrequency determined by the sum of all treatment groups of AR52Q flies.In FIG. 7D, the predicted population frequency of each treatment groupwas determined by the ethanol+DMSO group. In FIG. 7F, the predictedpopulation frequency of each treatment group was determined by the DHT+1μM ibuprofen group. *P≤0.05. All error bars represent s.e.m.

FIGS. 8A-8E demonstrate AF2 modulation rescues SBMA fly eye degenerationwithout reducing AR levels. FIG. 8A is representative images of SBMA flyeyes reared on food containing vehicle (EtOH), DHT, or DHT+bicalutamide(Bic), dimethylcurcumin (DMC), TA, or MEPB. Images were captured fromfour flies for each treatment group. FIG. 8B is images of representativeimmunoblot of AR expression in SBMA fly heads. FIG. 8C is a bar graphquantifying protein levels from three immunoblots, as depicted in FIG.8B. Data shown in FIG. 8C were evaluated by one-way ANOVA followed byDunnett posthoc analysis for pairwise comparisons between each treatmentgroup and the DHT-only group. *P≤0.05. All error bars represent s.e.m.FIG. 8D depicts flies expressing AR52Q in eyes were fed with drugs intheir adult stage for 4 days. FIG. 8E depicts flies expressing AR52Qwere raised on food containing drugs during their development from theirembryonic stage. Enclosed adults were immediately collected andprocessed. Three heads for each treatment were used to detectaggregation.

FIGS. 9A-9J demonstrate generation and characterization of a novel mousemodel of SBMA. FIG. 9A is a schematic depicting the cDNA construct usedto generate transgenic SBMA mice. FIG. 9B is an image of fluorescence insitu hybridization of an AR121Q-specific probe and a general marker ofchromosome 17 in chromosome spreads isolated from lungs of AR121Q mice.FIG. 9C is an image of western blot analysis of spinal cord and muscleexpression of transgenic human AR in AR121Q mice and a previouslypublished SBMA mouse model (AR97Q). FIG. 9D is a bar graph quantifyingthe levels of transgenic human AR compared with that of endogenous mouseAR in AR121Q and AR97Q mice. n=3 mice per group, *P≤0.05 by Studentt-test. FIGS. 9E-9G depict representative brain (FIG. 9E), testis (FIG.9F) and liver (FIG. 9G) sections from 7-week-old NTG and AR121Q mice.Sections were stained with H&E for assessment of morphology, in additionto AR (N20) and ubiquitin antibodies. Scale bars represent 100 μm. FIG.9H is images of PolyQ and ubiquitin costaining in skeletal muscle of NTGand AR121Q mice. FIG. 9I is bar graphs quantifying fibers withcentralized nuclei in lower and upper hindlimb muscles. n=2 to 3 miceper group, one-way ANOVA. FIG. 9J is representative images of testisfrom NTG, AR121Q and AR121Q treated with 100 mg/kg of MEPB mice. Scalebars represent 500 μm. All error bars represent s.e.m.

FIG. 10A and FIG. 10B demonstrate pharmacokinetics of MEPB and TA. FIG.10A is graphs of MEPB concentrations in plasma, liver, muscle, testes,brain, and spinal cord were measured at multiple time points (5 min to48 h) following a single intraperitoneal injection of 100 mg/kg bodyweight MEPB. MEPB half-life (T_(1/2)) was calculated for each tissue.Each square indicates one collected sample. FIG. 10B is a graph of TAconcentrations in plasma, liver, muscle, testes, brain, and spinal cordmeasured at multiple time points (1-48 h) following a singleintraperitoneal injection of 50 mg/kg body weight TA.

FIGS. 11A-111 demonstrate the effect of MEPB treatment on gait, claspingphenotype, muscle fiber type, and blood chemistry. FIG. 11A is images ofrepresentative footprint/gait analysis of 8-week-old AR121Q mice treatedwith MEPB. Hindpaws were painted blue and forepaws were painted red.FIG. 11B is video stills of clasping behavior in representative NTG andAR121Q mice. FIG. 11C and FIG. 11D are bar graphs quantifying the number(FIG. 11C) and size (FIG. 11D) of ChAT-positive motor neurons in theanterior horn of the thoracic spinal cord of NTG and AR121Q mice treatedwith vehicle, 50 mg/kg MEPB, or 100 mg/kg MEPB; n=2 to 3 mice pertreatment group. *P≤0.05 by one way ANOVA and Dunnett posthoc analysisfor pairwise comparing between each AR121Q treatment group and the NTGvehicle group. FIG. 11E and FIG. 11F are bar graphs quantifying meantype I and type II hindlimb muscle fiber staining in NTG and AR121Qmice, n=3 mice per treatment group. *P≤0.05 by two-way ANOVA and Dunnettposthoc analysis for pairwise comparisons between each treatment groupand their corresponding NTG control group. FIG. 11G is a bar graphquantifying mean blood concentrations of metabolites, electrolytes,enzymes, and other molecules associated with kidney and liver functionfrom NTG and AR121Q mice. Mean concentrations were normalized tovehicle-treated NTG mice in order to allow depiction of all chemistrieson one graph, n=2 mice per treatment group. Data were analyzed bytwo-way ANOVA FIG. 11H and FIG. 11I are bar graphs quantifying meanblood concentrations of serum creatine kinase (FIG. 11H) and serumtestosterone (FIG. 11I) in NTG and AR121Q mice treated with vehicle, 50mg/kg MEPB, or 100 mg/kg MEPB; n=3 mice per treatment group. Data wereevaluated by two-way ANOVA. All error bars represent s.e.m.

FIGS. 12A-12F demonstrate the effect of AF2 modulators on AR proteinlevels in MN1 and HEK293T cells. FIG. 12A is images of representativeimmunoblot of MN1 cells that were untransfected, stably transfected withAR24Q, or stably transfected with AR65Q. Cells were treated with vehicle(0.1% ethanol+0.1% DMSO), 10 nM DHT+0.1% DMSO, 10 nM DHT+10 μM TA, or 10nM DHT+10 μM MEPB for 24 h. Blots were stained with AR (D6F11) andtubulin antibodies. FIG. 12B is a bar graph quantifying AR proteinlevels from three immunoblots, as depicted in FIG. 12A. FIG. 12C isimages of representative immunoblot of stably transfected MN1-AR65Qcells treated with 10 nM DHT+TA or MEPB. FIG. 12D is a bar graphquantifying AR protein levels from three immunoblots, as depicted inFIG. 12C. FIG. 12E is images of representative immunoblot of HEK293Tcells transiently transfected with AR65Q for 24 h. Cells were treatedwith vehicle, 10 nM DHT, or 10 nM DHT+bicalutamide (Bic), TA, or MEPBfor 24 h, and blots (n=3) were stained with AR (D6F11) and tubulinantibodies. FIG. 12F is images of filter trap assay for AR aggregatesfrom lysates prepared from HEK293T cells transiently transfected withFLAG-AR65Q. Cellulose acetate membranes (n=3) were stained with FLAG(M2) antibody. All error bars represent s.e.m.

FIGS. 13A-13D demonstrate the effect of AF2 modulators on AR proteinlevels in AR121Q mice. FIG. 13A and FIG. 13C are images ofrepresentative immunoblot of skeletal muscle (FIG. 13A) and spinal cord(FIG. 13C) of AR121Q mice treated with vehicle, 50 mg/kg MEPB, or 100mg/kg MEPB. FIG. 13B and FIG. 13C are bar graphs quantifying mouse ARand human AR protein levels relative to REVERT total protein stain, n=3mice per treatment group. Data were analyzed by one-way ANOVA. All errorbars represent s.e.m.

DETAILED DESCRIPTION

In various aspects, pharmaceutical formulations and methods are providedfor treating spinobulbar muscular atrophy in a subject in need thereof.An effective amount of1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole(“MEPB”) has been found to ameliorate one or more symptoms ofspinobulbar muscular atrophy in the subject. For example, in someaspects an effective amount of MEPB has been found to prevent or delayloss of body weight, a loss of mobility, and/or a loss of physicalstrength in the subject. In some aspects an effective amount of MEPB hasbeen found to prevent or delay neurogenic atrophy and/or a loss ofspinal cord motor neurons in the subject. In some aspects an effectiveamount of MEPB has been found to restore frequency of type I myofibersto normal levels for a healthy subject and/or to reverse testicularatrophy in the subject.

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. The skilled artisan will recognize many variants andadaptations of the embodiments described herein. These variants andadaptations are intended to be included in the teachings of thisdisclosure and to be encompassed by the claims herein.

All publications and patents cited in this specification are cited todisclose and describe the methods and/or materials in connection withwhich the publications are cited. All such publications and patents areherein incorporated by references as if each individual publication orpatent were specifically and individually indicated to be incorporatedby reference. Such incorporation by reference is expressly limited tothe methods and/or materials described in the cited publications andpatents and does not extend to any lexicographical definitions from thecited publications and patents. Any lexicographical definition in thepublications and patents cited that is not also expressly repeated inthe instant specification should not be treated as such and should notbe read as defining any terms appearing in the accompanying claims. Thecitation of any publication is for its disclosure prior to the filingdate and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior disclosure. Further, the dates of publication provided could bedifferent from the actual publication dates that may need to beindependently confirmed.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of thepresent disclosure, the preferred methods and materials are nowdescribed. Functions or constructions well-known in the art may not bedescribed in detail for brevity and/or clarity. Embodiments of thepresent disclosure will employ, unless otherwise indicated, techniquesof anatomy, biochemistry, histology, microbiology, molecular biology,neuroscience, pharmacology, photobiology, physiology, toxicology, andthe like, which are within the skill of the art. Such techniques areexplained fully in the literature.

It should be noted that ratios, concentrations, amounts, and othernumerical data can be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a numerical range of “about 0.1%to about 5%” should be interpreted to include not only the explicitlyrecited values of about 0.1% to about 5%, but also include individualvalues (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%,2.2%, 3.3%, and 4.4%) within the indicated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the disclosure, e.g. thephrase “x to y” includes the range from ‘x’ to ‘y’ as well as the rangegreater than ‘x’ and less than ‘y’. The range can also be expressed asan upper limit, e.g. ‘about x, y, z, or less' and should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘less than x’, less than y’, and ‘less than z’.Likewise, the phrase ‘about x, y, z, or greater’ should be interpretedto include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ aswell as the ranges of ‘greater than x’, greater than y’, and ‘greaterthan z’. In some embodiments, the term “about” can include traditionalrounding according to significant figures of the numerical value. Inaddition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numericalvalues, includes “about ‘x’ to about ‘y’”.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. It will be further understoodthat terms, such as those defined in commonly used dictionaries, shouldbe interpreted as having a meaning that is consistent with their meaningin the context of the specification and relevant art and should not beinterpreted in an idealized or overly formal sense unless expresslydefined herein.

The articles “a” and “an,” as used herein, mean one or more when appliedto any feature in embodiments of the present invention described in thespecification and claims. The use of “a” and “an” does not limit themeaning to a single feature unless such a limit is specifically stated.The article “the” preceding singular or plural nouns or noun phrasesdenotes a particular specified feature or particular specified featuresand may have a singular or plural connotation depending upon the contextin which it is used.

The terms “subject” or “patient”, as used herein, refer to any organismto which the compounds may be administered, e.g., for experimental,therapeutic, diagnostic, and/or prophylactic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans) and/or plants. In certain aspects, the subject isa human.

The terms “treating” or “preventing”, as used herein, can includepreventing a disease, disorder or condition from occurring in an animalwhich may be predisposed to the disease, disorder and/or condition buthas not yet been diagnosed as having it; inhibiting the disease,disorder or condition, e.g., impeding its progress; and relieving thedisease, disorder, or condition, e.g., causing regression of thedisease, disorder and/or condition. Treating the disease, disorder, orcondition can include ameliorating at least one symptom of theparticular disease, disorder, or condition, even if the underlyingpathophysiology is not affected, such as treating the pain of a subjectby administration of an analgesic agent even though such agent does nottreat the cause of the pain.

The term “therapeutic effect” is art-recognized and refers to a local orsystemic effect in animals, particularly mammals, and more particularlyhumans caused by a pharmacologically active substance. The term thusmeans any substance intended for use in the diagnosis, cure, mitigation,treatment or prevention of disease or in the enhancement of desirablephysical or mental development and conditions in an animal or human.

The term “modulation” is art-recognized and refers to up regulation(i.e., activation or stimulation), down regulation (i.e., inhibition orsuppression) of a response, or the two in combination or apart.

“Parenteral administration”, as used herein, means administration by anymethod other than through the digestive tract or non-invasive topical orregional routes. For example, parenteral administration may includeadministration to a patient intravenously, intradermally,intraperitoneally, intrapleurally, intratracheally, intramuscularly,subcutaneously, subjunctivally, by injection, and by infusion.

“Enteral administration”, as used herein, means administration viaabsorption through the gastrointestinal tract. Enteral administrationcan include oral and sublingual administration, gastric administration,or rectal administration.

The terms “sufficient” and “effective”, as used interchangeably herein,refer to an amount (e.g., mass, volume, dosage, concentration, and/ortime period) needed to achieve one or more desired result(s). A“therapeutically effective amount” is at least the minimum concentrationrequired to effect a measurable improvement or prevention of any symptomor a particular condition or disorder, to effect a measurableenhancement of life expectancy, or to generally improve patient qualityof life. The therapeutically effective amount is thus dependent upon thespecific biologically active molecule and the specific condition ordisorder to be treated. Therapeutically effective amounts of many activeagents, such as antibodies, are well known in the art. Thetherapeutically effective amounts of anionic proteins, proteinanalogues, or nucleic acids hereinafter discovered or for treatingspecific disorders with known proteins, protein analogues, or nucleicacids to treat additional disorders may be determined by standardtechniques which are well within the craft of a skilled artisan, such asa physician.

For administration of a therapeutic composition as disclosed herein(e.g., a selective androgen receptor modulator, small moleculetherapeutic agent, or a salt or prodrug thereof), conventional methodsof extrapolating human dosage based on doses administered to a murineanimal model can be carried out using the conversion factor forconverting the mouse dosage to human dosage: Dose Human per kg=DoseMouse per kg×12 (Freireich, et al., (1966) Cancer Chemother Rep. 50:219-244). Doses can also be given in milligrams per square meter of bodysurface area because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions. Moreover,body surface area can be used as a common denominator for drug dosage inadults and children as well as in different animal species as describedby Freireich, et al. (Freireich et al., (1966) Cancer Chemother Rep.50:219-244). Briefly, to express a mg/kg dose in any given species asthe equivalent mg/m² dose, multiply the dose by the appropriate kmfactor. In an adult human, 100 mg/kg is equivalent to 100 mg/kg×37kg/m²=3700 mg/m².

The terms “bioactive agent” and “active agent”, as used interchangeablyherein, include, without limitation, physiologically orpharmacologically active substances that act locally or systemically inthe body. A bioactive agent is a substance used for the treatment (e.g.,therapeutic agent), prevention (e.g., prophylactic agent), diagnosis(e.g., diagnostic agent), cure or mitigation of disease or illness, asubstance which affects the structure or function of the body, orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

The term “prodrug” refers to an agent, including a nucleic acid orproteins that is converted into a biologically active form in vitroand/or in vivo. Prodrugs can be useful because, in some situations, theymay be easier to administer than the parent compound. For example, aprodrug may be bioavailable by oral administration whereas the parentcompound is not. The prodrug may also have improved solubility inpharmaceutical compositions compared to the parent drug. A prodrug maybe converted into the parent drug by various mechanisms, includingenzymatic processes and metabolic hydrolysis. Harper, N.J. (1962) DrugLatentiation in Jucker, ed. Progress in Drug Research, 4:221-294;Morozowich et al. (1977) Application of Physical Organic Principles toProdrug Design in E. B. Roche ed. Design of Biopharmaceutical Propertiesthrough Prodrugs and Analogs, APhA; Acad. Pharm. Sci.; E. B. Roche, ed.(1977) Bioreversible Carriers in Drug in Drug Design, Theory andApplication, APhA; H. Bundgaard, ed. (1985) Design of Prodrugs,Elsevier; Wang et al. (1999) Prodrug approaches to the improved deliveryof peptide drug, Curr. Pharm. Design. 5(4):265-287; Pauletti et al.(1997) Improvement in peptide bioavailability: Peptidomimetics andProdrug Strategies, Adv. Drug. Delivery Rev. 27:235-256; Mizen et al.(1998). The Use of Esters as Prodrugs for Oral Delivery of β-Lactamantibiotics, Pharm. Biotech. 11:345-365; Gaignault et al. (1996)Designing Prodrugs and Bioprecursors I. Carrier Prodrugs, Pract. Med.Chem. 671-696; M. Asgharnejad (2000). Improving Oral Drug Transport ViaProdrugs, in G. L. Amidon, P. I. Lee and E. M. Topp, Eds., TransportProcesses in Pharmaceutical Systems, Marcell Dekker, p. 185-218; Balantet al. (1990) Prodrugs for the improvement of drug absorption viadifferent routes of administration, Eur. J. Drug Metab. Pharmacokinet.,15(2): 143-53; Balimane and Sinko (1999). Involvement of multipletransporters in the oral absorption of nucleoside analogues, Adv. DrugDelivery Rev., 39(1-3):183-209; Browne (1997). Fosphenytoin (Cerebyx),Clin. Neuropharmacol. 20(1): 1-12; Bundgaard (1979). Bioreversiblederivatization of drugs—principle and applicability to improve thetherapeutic effects of drugs, Arch. Pharm. Chemi. 86(1): 1-39; H.Bundgaard, ed. (1985) Design of Prodrugs, New York: Elsevier; Fleisheret al. (1996) Improved oral drug delivery: solubility limitationsovercome by the use of prodrugs, Adv. Drug Delivery Rev. 19(2): 115-130;Fleisher et al. (1985) Design of prodrugs for improved gastrointestinalabsorption by intestinal enzyme targeting, Methods Enzymol. 112: 360-81;Farquhar D, et al. (1983) Biologically Reversible Phosphate-ProtectiveGroups, J. Pharm. Sci., 72(3): 324-325; Han, H. K. et al. (2000)Targeted prodrug design to optimize drug delivery, AAPS PharmSci., 2(1):E6; Sadzuka Y. (2000) Effective prodrug liposome and conversion toactive metabolite, Curr. Drug Metab., 1(1):31-48; D. M. Lambert (2000)Rationale and applications of lipids as prodrug carriers, Eur. J. Pharm.Sci., 11 Suppl. 2:S15-27; Wang, W. et al. (1999) Prodrug approaches tothe improved delivery of peptide drugs. Curr. Pharm. Des., 5(4):265-87.

The term “biocompatible”, as used herein, refers to a material thatalong with any metabolites or degradation products thereof that aregenerally non-toxic to the recipient and do not cause any significantadverse effects to the recipient. Generally speaking, biocompatiblematerials are materials which do not elicit a significant inflammatoryor immune response when administered to a patient.

The term “biodegradable” as used herein, generally refers to a materialthat will degrade or erode under physiologic conditions to smaller unitsor chemical species that are capable of being metabolized, eliminated,or excreted by the subject. The degradation time is a function ofcomposition and morphology. Degradation times can be from hours toweeks.

The term “pharmaceutically acceptable”, as used herein, refers tocompounds, materials, compositions, and/or dosage forms that are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio, in accordance withthe guidelines of agencies such as the U.S. Food and DrugAdministration. A “pharmaceutically acceptable carrier”, as used herein,refers to all components of a pharmaceutical formulation that facilitatethe delivery of the composition in vivo. Pharmaceutically acceptablecarriers include, but are not limited to, diluents, preservatives,binders, lubricants, disintegrators, swelling agents, fillers,stabilizers, and combinations thereof.

The term “molecular weight”, as used herein, generally refers to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (Mw) as opposed to the number-average molecular weight(M). Capillary viscometry provides estimates of molecular weight as theinherent viscosity determined from a dilute polymer solution using aparticular set of concentration, temperature, and solvent conditions.

The term “small molecule”, as used herein, generally refers to anorganic molecule that is less than 2000 g/mol in molecular weight, lessthan 1500 g/mol, less than 1000 g/mol, less than 800 g/mol, or less than500 g/mol. Small molecules are non-polymeric and/or non-oligomeric.

The term “hydrophilic”, as used herein, refers to substances that havestrongly polar groups that readily interact with water.

The term “hydrophobic”, as used herein, refers to substances that lackan affinity for water; tending to repel and not absorb water as well asnot dissolve in or mix with water.

The term “lipophilic”, as used herein, refers to compounds having anaffinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combininghydrophilic and lipophilic (hydrophobic) properties. “Amphiphilicmaterial” as used herein refers to a material containing a hydrophobicor more hydrophobic oligomer or polymer (e.g., biodegradable oligomer orpolymer) and a hydrophilic or more hydrophilic oligomer or polymer.

The term “activated ester”, as used herein, refers to alkyl esters ofcarboxylic acids where the alkyl is a good leaving group rendering thecarbonyl susceptible to nucleophilic attack by molecules bearing aminogroups. Activated esters are therefore susceptible to aminolysis andreact with amines to form amides. Activated esters contain a carboxylicacid ester group —CO₂R where R is the leaving group.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In some embodiments, a straight chain or branched chain alkyl has 30 orfewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,C₃-C₃₀ for branched chains), 20 or fewer, 12 or fewer, or 7 or fewer.Likewise, in some embodiments cycloalkyls have from 3-10 carbon atoms intheir ring structure, e.g. have 5, 6 or 7 carbons in the ring structure.The term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having one or more substituents replacing ahydrogen on one or more carbons of the hydrocarbon backbone. Suchsubstituents include, but are not limited to, halogen, hydroxyl,carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl),thiocarbonyl (such as a thioester, a thioacetate, or a thioformate),alkoxyl, phosphoryl, phosphate, phosphonate, a hosphinate, amino, amido,amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate,sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, oran aromatic or heteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, or from one to six carbon atoms in its backbonestructure. Likewise, “lower alkenyl” and “lower alkynyl” have similarchain lengths. Throughout the application, alkyl groups can be loweralkyls. In some embodiments, a substituent designated herein as alkyl isa lower alkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, hydroxy, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF₃, —CN and the like. Cycloalkyls can besubstituted in the same manner.

The term “heteroalkyl”, as used herein, refers to straight or branchedchain, or cyclic carbon-containing radicals, or combinations thereof,containing at least one heteroatom. Suitable heteroatoms include, butare not limited to, O, N, Si, P, Se, B, and S, wherein the phosphorousand sulfur atoms are optionally oxidized, and the nitrogen heteroatom isoptionally quaternized. Heteroalkyls can be substituted as defined abovefor alkyl groups.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In some embodiments, the “alkylthio”moiety is represented by one of —S-alkyl, —S— alkenyl, and —S-alkynyl.Representative alkylthio groups include methylthio, and ethylthio. Theterm “alkylthio” also encompasses cycloalkyl groups, alkene andcycloalkene groups, and alkyne groups. “Arylthio” refers to aryl orheteroaryl groups. Alkylthio groups can be substituted as defined abovefor alkyl groups.

The terms “alkenyl” and “alkynyl”, refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy, andtert-butoxy. An “ether” is two hydrocarbons covalently linked by anoxygen. Accordingly, the substituent of an alkyl that renders that alkylan ether is or resembles an alkoxyl, such as can be represented by oneof —O-alkyl, —O-alkenyl, and —O-alkynyl. Aroxy can be represented by—O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as definedbelow. The alkoxy and aroxy groups can be substituted as described abovefor alkyl.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₈ or R₉ and R₁₀ taken together with the Natom to which they are attached complete a heterocycle having from 4 to8 atoms in the ring structure; R₃ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In some embodiments, only one of R₉ or R₁₀ canbe a carbonyl, e.g., R₉, R₁₀ and the nitrogen together do not form animide. In still other embodiments, the term “amine” does not encompassamides, e.g., wherein one of R₉ and R₁₀ represents a carbonyl. Inadditional embodiments, R₉ and R₁₀ (and optionally R′₁₀) eachindependently represent a hydrogen, an alkyl or cycloakly, an alkenyl orcycloalkenyl, or alkynyl. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted (asdescribed above for alkyl) or unsubstituted alkyl attached thereto,i.e., at least one of R₉ and R₁₀ is an alkyl group.

The term “amido” is art-recognized as an amino-substituted carbonyl andincludes a moiety that can be represented by the general formula:

wherein R₉ and R₁₀ are as defined above.

“Aryl”, as used herein, refers to C₅-C₁₀-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl,imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl,3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl,isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl,methylenedioxyphenyl, morpholinyl, naphthyridinyl,octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “carbocycle”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

“Heterocycle” or “heterocyclic”, as used herein, refers to a cyclicradical attached via a ring carbon or nitrogen of a monocyclic orbicyclic ring containing 3-10 ring atoms, or from 5-6 ring atoms,consisting of carbon and one to four heteroatoms each selected from thegroup consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y isabsent or is H, O, (C₁-C₁₀) alkyl, phenyl or benzyl, and optionallycontaining 1-3 double bonds and optionally substituted with one or moresubstituents. Examples of heterocyclic ring include, but are not limitedto, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl,benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl,carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl,cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxepanyl, oxetanyl, oxindolyl, pyrimidinyl, phenanthridinyl,phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl,phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl,4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl,pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl,pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl,quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydropyranyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. Heterocyclicgroups can optionally be substituted with one or more substituents atone or more positions as defined above for alkyl and aryl, for example,halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF3, and—CN.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, a cycloalkyl, an alkenyl, ancycloalkenyl, or an alkynyl, R′₁₁ represents a hydrogen, an alkyl, acycloalkyl, an alkenyl, an cycloalkenyl, or an alkynyl. Where X is anoxygen and R₁₁ or R′₁₁ is not hydrogen, the formula represents an“ester”. Where X is an oxygen and R₁₁ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₁₁ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen and R′₁₁ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R′₁₁ ishydrogen, the formula represents a “thioformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “monoester” as used herein refers to an analogue of adicarboxylic acid wherein one of the carboxylic acids is functionalizedas an ester and the other carboxylic acid is a free carboxylic acid orsalt of a carboxylic acid. Examples of monoesters include, but are notlimited to, to monoesters of succinic acid, glutaric acid, adipic acid,suberic acid, sebacic acid, azelaic acid, oxalic and maleic acid.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Examples of heteroatoms are boron, nitrogen,oxygen, phosphorus, sulfur and selenium. Other heteroatoms includesilicon and arsenic.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “substituted” as used herein, refers to all permissiblesubstituents of the compounds described herein. In the broadest sense,the permissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,but are not limited to, halogens, hydroxyl groups, or any other organicgroupings containing any number of carbon atoms, e.g. 1-14 carbon atoms,and optionally include one or more heteroatoms such as oxygen, sulfur,or nitrogen grouping in linear, branched, or cyclic structural formats.Representative substituents include alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substitutedphenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl,halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substitutedphenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio,phenylthio, substituted phenylthio, arylthio, substituted arylthio,cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl,carboxyl, substituted carboxyl, amino, substituted amino, amido,substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid,phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl,polyaryl, substituted polyaryl, C₃-C₂₀ cyclic, substituted C₃-C₂₀cyclic, heterocyclic, substituted heterocyclic, aminoacid, peptide, andpolypeptide groups.

Heteroatoms such as nitrogen may have hydrogen substituents and/or anypermissible substituents of organic compounds described herein whichsatisfy the valences of the heteroatoms. It is understood that“substitution” or “substituted” includes the implicit proviso that suchsubstitution is in accordance with permitted valence of the substitutedatom and the substituent, and that the substitution results in a stablecompound, i.e. a compound that does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.

In a broad aspect, the permissible substituents include acyclic andcyclic, branched and unbranched, carbocyclic and heterocyclic, aromaticand nonaromatic substituents of organic compounds. Illustrativesubstituents include, for example, those described herein. Thepermissible substituents can be one or more and the same or differentfor appropriate organic compounds. The heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms.

In various embodiments, the substituent is selected from alkoxy,aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen,haloalkyl, heteroaryl, heterocyclyl, hydroxyl, ketone, nitro, phosphate,sulfide, sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone,each of which optionally is substituted with one or more suitablesubstituents. In some embodiments, the substituent is selected fromalkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl,carbamate, carboxy, cycloalkyl, ester, ether, formyl, haloalkyl,heteroaryl, heterocyclyl, ketone, phosphate, sulfide, sulfinyl,sulfonyl, sulfonic acid, sulfonamide, and thioketone, wherein each ofthe alkoxy, aryloxy, alkyl, alkenyl, alkynyl, amide, amino, aryl,arylalkyl, carbamate, carboxy, cycloalkyl, ester, ether, formyl,haloalkyl, heteroaryl, heterocyclyl, ketone, phosphate, sulfide,sulfinyl, sulfonyl, sulfonic acid, sulfonamide, and thioketone can befurther substituted with one or more suitable substituents.

Examples of substituents include, but are not limited to, halogen,azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl,amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido,ketone, aldehyde, thioketone, ester, heterocyclyl, —CN, aryl, aryloxy,perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl,heteroaralkoxy, azido, alkylthio, oxo, acylalkyl, carboxy esters,carboxamido, acyloxy, aminoalkyl, alkylaminoaryl, alkylaryl,alkylaminoalkyl, alkoxyaryl, arylamino, aralkylamino, alkylsulfonyl,carboxamidoalkylaryl, carboxamidoaryl, hydroxyalkyl, haloalkyl,alkylaminoalkylcarboxy, aminocarboxamidoalkyl, cyano, alkoxyalkyl,perhaloalkyl, arylalkyloxyalkyl, and the like. In some embodiments, thesubstituent is selected from cyano, halogen, hydroxyl, and nitro.

The term “copolymer” as used herein, generally refers to a singlepolymeric material that is comprised of two or more different monomers.The copolymer can be of any form, such as random, block, graft, etc. Thecopolymers can have any end-group, including capped or acid end groups.

The terms “polypeptide,” “peptide” and “protein” generally refer to apolymer of amino acid residues. As used herein, the term also applies toamino acid polymers in which one or more amino acids are chemicalanalogues or modified derivatives of corresponding naturally-occurringamino acids. The term “protein”, as generally used herein, refers to apolymer of amino acids linked to each other by peptide bonds to form apolypeptide for which the chain length is sufficient to produce tertiaryand/or quaternary structure. The term “protein” excludes small peptidesby definition, the small peptides lacking the requisite higher-orderstructure necessary to be considered a protein.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably to refer to a deoxyribonucleotide or ribonucleotidepolymer, in linear or circular conformation, and in either single- ordouble-stranded form. These terms are not to be construed as limitingwith respect to the length of a polymer. The terms can encompass knownanalogues of natural nucleotides, as well as nucleotides that aremodified in the base, sugar and/or phosphate moieties (e.g.,phosphorothioate backbones). In general and unless otherwise specified,an analogue of a particular nucleotide has the same base-pairingspecificity; i.e., an analogue of A will base-pair with T. The term“nucleic acid” is a term of art that refers to a string of at least twobase-sugar-phosphate monomeric units. Nucleotides are the monomericunits of nucleic acid polymers. The term includes deoxyribonucleic acid(DNA) and ribonucleic acid (RNA) in the form of a messenger RNA,antisense, plasmid DNA, parts of a plasmid DNA or genetic materialderived from a virus. Antisense is a polynucleotide that interferes withthe function of DNA and/or RNA. The term nucleic acids refers to astring of at least two base-sugar-phosphate combinations. Naturalnucleic acids have a phosphate backbone, artificial nucleic acids maycontain other types of backbones, but contain the same bases. The termalso includes PNAs (peptide nucleic acids), phosphorothioates, and othervariants of the phosphate backbone of native nucleic acids.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains atleast one function as the full-length protein, polypeptide or nucleicacid. A functional fragment can possess more, fewer, or the same numberof residues as the corresponding native molecule, and/or can contain oneor more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. The ability of a protein to interact with anotherprotein can be determined, for example, by co-immunoprecipitation,two-hybrid assays or complementation, e.g., genetic or biochemical. See,for example, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

As used herein, the term “linker” refers to a carbon chain that cancontain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. Linkersmay be substituted with various substituents including, but not limitedto, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, thioether, alkylthioether, thiol, and ureido groups. Thoseof skill in the art will recognize that each of these groups may in turnbe substituted. Examples of linkers include, but are not limited to,pH-sensitive linkers, protease cleavable peptide linkers, nucleasesensitive nucleic acid linkers, lipase sensitive lipid linkers,glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers,photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers(e.g., esterase cleavable linker), ultrasound-sensitive linkers, andx-ray cleavable linkers.

The term “pharmaceutically acceptable counter ion” refers to apharmaceutically acceptable anion or cation. In various embodiments, thepharmaceutically acceptable counter ion is a pharmaceutically acceptableion. For example, the pharmaceutically acceptable counter ion isselected from citrate, matate, acetate, oxalate, chloride, bromide,iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, tartrate, oleate, tannate,pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate,fumarate, gluconate, glucaronate, saccharate, formate, benzoate,glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)). In some embodiments, thepharmaceutically acceptable counter ion is selected from chloride,bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,citrate, malate, acetate, oxalate, acetate, and lactate. In particularembodiments, the pharmaceutically acceptable counter ion is selectedfrom chloride, bromide, iodide, nitrate, sulfate, bisulfate, andphosphate.

The term “pharmaceutically acceptable salt(s)” refers to salts of acidicor basic groups that may be present in compounds used in the presentcompositions. Compounds included in the present compositions that arebasic in nature are capable of forming a wide variety of salts withvarious inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to sulfate, citrate, matate, acetate, oxalate, chloride,bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate,isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate,tannate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate,benzenesulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds includedin the present compositions that include an amino moiety may formpharmaceutically acceptable salts with various amino acids, in additionto the acids mentioned above. Compounds included in the presentcompositions, that are acidic in nature are capable of forming basesalts with various pharmacologically acceptable cations. Examples ofsuch salts include alkali metal or alkaline earth metal salts and,particularly, calcium, magnesium, sodium, lithium, zinc, potassium, andiron salts.

If the compounds described herein are obtained as an acid addition salt,the free base can be obtained by basifying a solution of the acid salt.Conversely, if the product is a free base, an addition salt,particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare non-toxic pharmaceutically acceptable additionsalts.

A pharmaceutically acceptable salt can be derived from an acid selectedfrom 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid,2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoicacid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid,aspartic acid, benzenesulfonic acid, benzoic acid, camphoric acid,camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid(hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamicacid, citric acid, cyclamic acid, dodecylsulfuric acid,ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaricacid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid,glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid,glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid,isethionic, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonicacid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmiticacid, pamoic acid, pantothenic, phosphoric acid, proprionic acid,pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinicacid, sulfuric acid, tartaric acid, thiocyanic acid, toluenesulfonicacid, trifluoroacetic, and undecylenic acid.

The term “bioavailable” is art-recognized and refers to a form of thesubject invention that allows for it, or a portion of the amountadministered, to be absorbed by, incorporated to, or otherwisephysiologically available to a subject or patient to whom it isadministered.

Pharmaceutical Formulations

A variety of pharmaceutical formulations are provided for treatingspinobulbar muscular atrophy in a subject in need thereof. In someaspects, the formulations contain an effective amount of a selectiveandrogen receptor modulator. In some aspects, the formulations containan effective amount of a small molecule having a structure according tothe following formula:

In the above formula, each occurrence of R¹, R², R³, and R⁴ can bechosen independently to be a suitable substituent. In some aspects, eachoccurrence of R¹, R², R³, and R⁴ is independently a hydrogen, ahydroxyl, a halogen, or a substituted or unsubstituted C₁-C₁₂, C₁-C₁₀,C₁-C₆, C₂-C₁₂, or C₂-C₆ alkyl or alkoxy. In the above formula, eachoccurrence of X¹ and X² can be independently a heteroatom, e.g. O or S.In the above formula, each occurrence of A¹ is independently none (i.e.is a bond) or is a substituted or unsubstituted C₁-C₁₂, C₁-C₁₀, C₁-C₆,C₂-C₁₂, or C₂-C₆ alkyl.

The formulations can contain an effective amount of a selective androgenreceptor modulator. The term “selective androgen receptor modulator,” asused herein, refers to a class of androgen receptor ligands that exhibittheir activity in a tissue selective manner. In other words, tissueselectivity allows a nuclear receptor ligand to function as an agonistin some tissues, while having no effect or even an antagonist effect inother tissues. A synthetic compound that binds to an intracellularreceptor and mimics the effects of the native hormone is referred to asan agonist. A compound that inhibits the effect of the native hormone iscalled an antagonist. The term “modulators” can refer to compounds thathave a spectrum of activities ranging from full agonism to partialagonism to full antagonism.

The selective androgen receptor modulator can selectively bind to theandrogen receptor. In some aspects, the selective androgen receptormodulator can selectively bind to the binding function-3 (BF3) domain ofthe androgen receptor.

In some aspects, the selective androgen receptor modulator alters aco-regulator binding to the activation function-2 (AF2) domain of theandrogen receptor. For example, the selective androgen receptormodulator can partially or completely inhibit co-regulator binding tothe activation function-2 (AF2) domain of the androgen receptor.

The term “selectively binds” refers to the ability of a selectivebinding compound to bind to a target receptor or to a specific bindingdomain with greater affinity than it binds to a non-target receptor orbinding domain. In certain aspects, selective binding refers to bindingto a target with an affinity that is at least 10, 50, 100, 250, 500, or1000 times greater than the affinity for a non-target. The term “targetreceptor” refers to a receptor or a portion of a receptor capable ofbeing bound by a selective binding compound. In certain aspects, atarget receptor is an androgen receptor, and in particular in certainaspects the target receptor is the binding function-3 (BF3) domain ofthe androgen receptor.

In some aspects, the selective androgen receptor modulator is a smallmolecule described herein. In some aspects, the selective androgenreceptor modulator is 17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one ora derivative thereof. 17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one,also known as Oxandrolone, is marketed under the brand names OXANDRIN byGemini Laboratories (NJ, USA). In some aspects, the selective androgenreceptor modulator is testosterone or a derivative thereof. In someaspects, the selective androgen receptor is a testosterone ester such astestosterone enanthate, testosterone propionate, or testosteronecypionate. In some aspects, the selective androgen receptor modulator is4,5α-dihydrotestosterone or a derivative thereof. In some aspects, theselective androgen receptor modulator is((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide)or a derivative thereof.((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide),also known as ostarine or enobosarm, is an investigational selectiveandrogen receptor developed by Merck and Company (NJ, USA).

In some aspects, the pharmaceutical formulation includes a derivative ofthe small molecule or a derivative of a selective androgen receptormodulator described herein. Suitable derivatives can include ester andamide derivatives, pegylated derivatives, and N-oxides. The derivativecan include replacing or substituting one of more R groups with asubstituent such as a substituent selected from the group consisting ofa halogen, an azide, an alkyl, an aralkyl, an alkenyl, an alkynyl, acycloalkyl, a hydroxyl, an alkoxyl, an amino, a nitro, a sulfhydryl, animino, an amido, a phosphonate, a phosphinate, a carbonyl, a carboxyl, asilyl, an ether, an alkylthio, a sulfonyl, a sulfonamido, a ketone, analdehyde, a thioketone, an ester, a heterocyclyl, a —CN, an aryl, anaryloxy, a perhaloalkoxy, an aralkoxy, a heteroaryl, a heteroaryloxy, aheteroarylalkyl, a heteroaralkoxy, an azido, an alkylthio, an oxo, anacylalkyl, a carboxy esters, a carboxamido, an acyloxy, an aminoalkyl,an alkylaminoaryl, an alkylaryl, an alkylaminoalkyl, an alkoxyaryl, anarylamino, an aralkylamino, an alkylsulfonyl, a carboxamidoalkylaryl, acarboxamidoaryl, a hydroxyalkyl, a haloalkyl, an alkylaminoalkylcarboxy,an aminocarboxamidoalkyl, a cyano, an alkoxyalkyl, a perhaloalkyl, andan arylalkyloxyalkyl.

In some aspects, the pharmaceutical formulation includes a prodrug ofthe small molecule or a derivative thereof. Methods of making prodrugsare known in the art, and can include an amide, carbamate, imide, ester,anhydride, thioester, or thioanhydride of the small molecule orderivative thereof.

In some aspects, the pharmaceutical formulation includes a salt of thesmall molecule or a derivative or prodrug thereof. The salt can be apharmaceutically acceptable acid addition salt including an anion suchas a sulfate, a citrate, matate, an acetate, an oxalate, a chloride, abromide, an iodide, a nitrate, a sulfate, a bisulfate, a phosphate, anacid phosphate, an isonicotinate, an acetate, a lactate, a salicylate, atartrate, an oleate, a tannate, a pantothenate, a bitartrate, anascorbate, a succinate, a maleate, a gentisinate, a fumarate, agluconate, a glucaronate, a saccharate, a formate, a benzoate, aglutamate, a methanesulfonate, an ethanesulfonate, a benzenesulfonate, ap-toluenesulfonate or a pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)). The salt can be a baseaddition salt including an alkali metal and alkaline earth metal cation,such as calcium, magnesium, sodium, lithium, zinc, potassium, or iron.

The term “pharmaceutically acceptable salts”, as used herein, meanssalts of the active principal agents which are prepared with acids orbases that are tolerated by a biological system or tolerated by asubject or tolerated by a biological system and tolerated by a subjectwhen administered in a therapeutically effective amount. When compoundsof the present disclosure contain relatively acidic functionalities,base addition salts can be obtained by contacting the neutral form ofsuch compounds with a sufficient amount of the desired base, either neator in a suitable inert solvent. Examples of pharmaceutically acceptablebase addition salts include, but are not limited to; sodium, potassium,calcium, ammonium, organic amino, magnesium salt, lithium salt,strontium salt or a similar salt. When compounds of the presentdisclosure contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include, but are not limited to; those derived from inorganicacids like hydrochloric, hydrobromic, nitric, carbonic,monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived fromrelatively nontoxic organic acids like acetic, propionic, isobutyric,maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic,phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric,methanesulfonic, and the like. Also included are salts of amino acidssuch as arginate and the like, and salts of organic acids likeglucuronic or galactunoric acids and the like.

The term “pharmaceutically acceptable ester” refers to esters ofcompounds of the present disclosure which hydrolyze in vivo and includethose that break down readily in the human body to leave the parentcompound or a salt thereof. Examples of pharmaceutically acceptable,non-toxic esters of the present disclosure include C 1-to-C 6 alkylesters and C 5-to-C 7 cycloalkyl esters, although C 1-to-C 4 alkylesters are preferred. Esters of disclosed compounds can be preparedaccording to conventional methods. Pharmaceutically acceptable esterscan be appended onto hydroxy groups by reaction of the compound thatcontains the hydroxy group with acid and an alkylcarboxylic acid such asacetic acid, or with acid and an arylcarboxylic acid such as benzoicacid. In the case of compounds containing carboxylic acid groups, thepharmaceutically acceptable esters are prepared from compoundscontaining the carboxylic acid groups by reaction of the compound withbase such as triethylamine and an alkyl halide, for example with methyliodide, benzyl iodide, cyclopentyl iodide or alkyl triflate. They alsocan be prepared by reaction of the compound with an acid such ashydrochloric acid and an alcohol such as ethanol or methanol.

The term “pharmaceutically acceptable amide” refers to non-toxic amidesof the present disclosure derived from ammonia, primary C 1-to-C 6 alkylamines and secondary C 1-to-C 6 dialkyl amines. In the case of secondaryamines, the amine can also be in the form of a 5- or 6-memberedheterocycle containing one nitrogen atom. Amides derived from ammonia, C1-to-C 3 alkyl primary amides and C 1-to-C 2 dialkyl secondary amidesare preferred. Amides of disclosed compounds can be prepared accordingto conventional methods. Pharmaceutically acceptable amides can beprepared from compounds containing primary or secondary amine groups byreaction of the compound that contains the amino group with an alkylanhydride, aryl anhydride, acyl halide, or aroyl halide. In the case ofcompounds containing carboxylic acid groups, the pharmaceuticallyacceptable amides are prepared from compounds containing the carboxylicacid groups by reaction of the compound with base such as triethylamine,a dehydrating agent such as dicyclohexyl carbodiimide or carbonyldiimidazole, and an alkyl amine, dialkylamine, for example withmethylamine, diethylamine, and piperidine. They also can be prepared byreaction of the compound with an acid such as sulfuric acid and analkylcarboxylic acid such as acetic acid, or with acid and anarylcarboxylic acid such as benzoic acid under dehydrating conditionssuch as with molecular sieves added. The composition can contain acompound of the present disclosure in the form of a pharmaceuticallyacceptable prodrug.

The term “pharmaceutically acceptable prodrug” or “prodrug” representsthose prodrugs of the compounds of the present disclosure which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use.Prodrugs of the present disclosure can be rapidly transformed in vivo toa parent compound having a structure of a disclosed compound, forexample, by hydrolysis in blood. A thorough discussion is provided in T.Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of theA.C.S. Symposium Series, and in Edward B. Roche, ed., BioreversibleCarriers in Drug Design, American Pharmaceutical Association andPergamon Press (1987).

In some aspects, the small molecule is-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazolehaving a structure according to the following formula

The formulations include a therapeutically effective amount of the smallmolecule or a selective androgen receptor modulator or a derivative,prodrug, or salt thereof. In some aspects, the therapeutically effectiveamount is effective to prevent or delay loss of body weight, a loss ofmobility, and/or a loss of physical strength in the subject. In someaspects, the therapeutically effective amount is effective to prevent ordelay neurogenic atrophy and/or to prevent a loss of spinal cord motorneurons in the subject. In some aspects, the therapeutically effectiveamount is effective to restore the frequency of type I myofibers tonormal levels for a healthy subject. In some aspects, thetherapeutically effective amount is effective to reverse testicularatrophy in the subject.

Parenteral Formulations

The small molecules or a derivative, prodrug, or salt thereof can beformulated for parenteral delivery, such as injection or infusion, inthe form of a solution or suspension. The formulation can beadministered via any route, such as, the blood stream or directly to theorgan or tissue to be treated.

Parenteral formulations can be prepared as aqueous compositions usingtechniques is known in the art. Typically, such compositions can beprepared as injectable formulations, for example, solutions orsuspensions; solid forms suitable for using to prepare solutions orsuspensions upon the addition of a reconstitution medium prior toinjection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water(o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, one or more polyols (e.g., glycerol, propyleneglycol, and liquid polyethylene glycol), oils, such as vegetable oils(e.g., peanut oil, corn oil, sesame oil, etc.), and combinationsthereof. The proper fluidity can be maintained, for example, by the useof a coating, such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and/or by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride.

Solutions and dispersions of the small molecules can be prepared inwater or another solvent or dispersing medium suitably mixed with one ormore pharmaceutically acceptable excipients including, but not limitedto, surfactants, dispersants, emulsifiers, pH modifying agents, andcombination thereof.

Suitable surfactants may be anionic, cationic, amphoteric or nonionicsurface active agents. Suitable anionic surfactants include, but are notlimited to, those containing carboxylate, sulfonate and sulfate ions.Examples of anionic surfactants include sodium, potassium, ammonium oflong chain alkyl sulfonates and alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumdodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-p-alanine, sodium N-lauryl-p-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

The formulation can contain a preservative to prevent the growth ofmicroorganisms. Suitable preservatives include, but are not limited to,parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. Theformulation may also contain an antioxidant to prevent degradation ofthe small molecule(s) or active agent(s).

The formulation is typically buffered to a pH of 3-8 for parenteraladministration upon reconstitution. Suitable buffers include, but arenot limited to, phosphate buffers, acetate buffers, and citrate buffers.

Water soluble polymers are often used in formulations for parenteraladministration. Suitable water-soluble polymers include, but are notlimited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, andpolyethylene glycol.

Sterile injectable solutions can be prepared by incorporating the smallmolecules in the required amount in the appropriate solvent ordispersion medium with one or more of the excipients listed above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the various sterilized small molecules into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those listed above. In the case ofsterile powders for the preparation of sterile injectable solutions, themethods of preparation can include vacuum-drying and freeze-dryingtechniques which yield a powder of the small molecules plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The powders can be prepared in such a manner that theparticles are porous in nature, which can increase dissolution of theparticles. Methods for making porous particles are well known in theart.

Pharmaceutical formulations for parenteral administration can be in theform of a sterile aqueous solution or suspension of particles formedfrom one or more small molecules. Acceptable solvents include, forexample, water, Ringer's solution, phosphate buffered saline (PBS), andisotonic sodium chloride solution. The formulation may also be a sterilesolution, suspension, or emulsion in a nontoxic, parenterally acceptablediluent or solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in aliquid form. Alternatively, formulations for parenteral administrationcan be packed as a solid, obtained, for example by lyophilization of asuitable liquid formulation. The solid can be reconstituted with anappropriate carrier or diluent prior to administration.

Solutions, suspensions, or emulsions for parenteral administration maybe buffered with an effective amount of buffer necessary to maintain apH suitable for ocular administration. Suitable buffers are well knownby those skilled in the art and some examples of useful buffers areacetate, borate, carbonate, citrate, and phosphate buffers.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more tonicity agents to adjust the isotonic range ofthe formulation. Suitable tonicity agents are well known in the art andsome examples include glycerin, mannitol, sorbitol, sodium chloride, andother electrolytes.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more preservatives to prevent bacterialcontamination of the ophthalmic preparations. Suitable preservatives areknown in the art, and include polyhexamethylenebiguanidine (PHMB),benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwiseknown as Purite®), phenylmercuric acetate, chlorobutanol, sorbic acid,chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixturesthereof.

Solutions, suspensions, or emulsions for parenteral administration mayalso contain one or more excipients known art, such as dispersingagents, wetting agents, and suspending agents.

Enteral Formulations

The small molecules or a derivative, prodrug, or salt thereof can beprepared in enteral formulations, such as for oral administration.Suitable oral dosage forms include tablets, capsules, solutions,suspensions, syrups, and lozenges. Tablets can be made using compressionor molding techniques well known in the art. Gelatin or non-gelatincapsules can prepared as hard or soft capsule shells, which canencapsulate liquid, solid, and semi-solid fill materials, usingtechniques well known in the art.

Formulations are prepared using pharmaceutically acceptable carriers. Asgenerally used herein “carrier” includes, but is not limited to,diluents, preservatives, binders, lubricants, disintegrators, swellingagents, fillers, stabilizers, and combinations thereof. Polymers used inthe dosage form include hydrophobic or hydrophilic polymers and pHdependent or independent polymers. Preferred hydrophobic and hydrophilicpolymers include, but are not limited to, hydroxypropyl methylcellulose,hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethylcellulose, polyethylene glycol, ethylcellulose, microcrystallinecellulose, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl acetate,and ion exchange resins.

Carrier also includes all components of the coating composition whichmay include plasticizers, pigments, colorants, stabilizing agents, andglidants.

Formulations can be prepared using one or more pharmaceuticallyacceptable excipients, including diluents, preservatives, binders,lubricants, disintegrators, swelling agents, fillers, stabilizers, andcombinations thereof.

Delayed release dosage formulations can be prepared as described instandard references such as “Pharmaceutical dosage form tablets”, eds.Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington—Thescience and practice of pharmacy”, 20th ed., Lippincott Williams &Wilkins, Baltimore, M D, 2000, and “Pharmaceutical dosage forms and drugdelivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams andWilkins, 1995). These references provide information on excipients,materials, equipment and process for preparing tablets and capsules anddelayed release dosage forms of tablets, capsules, and granules. Thesereferences provide information on carriers, materials, equipment andprocess for preparing tablets and capsules and delayed release dosageforms of tablets, capsules, and granules.

The small molecules can be formulated in nanoparticles or in variouscapsule forms which may be coated, for example to delay release whenpassing through the acidic environment of the stomach. Examples ofsuitable coating materials include, but are not limited to, cellulosepolymers such as cellulose acetate phthalate, hydroxypropyl cellulose,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalateand hydroxypropyl methylcellulose acetate succinate; polyvinyl acetatephthalate, acrylic acid polymers and copolymers, and methacrylic resinsthat are commercially available under the trade name EUDRAGIT® (RothPharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Coatings may be formed with a different ratio of water soluble polymer,water insoluble polymers and/or pH dependent polymers, with or withoutwater insoluble/water soluble non polymeric excipient, to produce thedesired release profile. The coating is either performed on dosage form(matrix or simple) which includes, but not limited to, tablets(compressed with or without coated beads), capsules (with or withoutcoated beads), beads, particle compositions, “ingredient as is”formulated as, but not limited to, suspension form or as a sprinkledosage form.

Examples of suitable coating materials include, but are not limited to,cellulose polymers such as cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulosephthalate and hydroxypropyl methylcellulose acetate succinate; polyvinylacetate phthalate, acrylic acid polymers and copolymers, and methacrylicresins that are commercially available under the trade name EUDRAGIT®(Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.

Additionally, the coating material may contain conventional carrierssuch as plasticizers, pigments, colorants, glidants, stabilizationagents, pore formers and surfactants.

Optional pharmaceutically acceptable excipients include, but are notlimited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (Polyplasdone® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard drug decomposition reactionswhich include, by way of example, oxidative reactions. Suitablestabilizers include, but are not limited to, antioxidants, butylatedhydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E,tocopherol and its salts; sulfites such as sodium metabisulphite;cysteine and its derivatives; citric acid; propyl gallate, and butylatedhydroxyanisole (BHA).

Diluents, also referred to as “fillers,” are typically necessary toincrease the bulk of a solid dosage form so that a practical size isprovided for compression of tablets or formation of beads and granules.Suitable diluents include, but are not limited to, dicalcium phosphatedihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol,cellulose, microcrystalline cellulose, kaolin, sodium chloride, drystarch, hydrolyzed starches, pregelatinized starch, silicone dioxide,titanium oxide, magnesium aluminum silicate and powdered sugar. Theusual diluents include inert powdered substances such as starches,powdered cellulose, especially crystalline and microcrystallinecellulose, sugars such as fructose, mannitol and sucrose, grain floursand similar edible powders. Typical diluents include, for example,various types of starch, lactose, mannitol, kaolin, calcium phosphate orsulfate, inorganic salts such as sodium chloride and powdered sugar.Powdered cellulose derivatives are also useful. Typical tablet bindersinclude substances such as starch, gelatin and sugars such as lactose,fructose, and glucose. Natural and synthetic gums, including acacia,alginates, methylcellulose, and polyvinylpyrrolidone can also be used.Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes canalso serve as binders. A lubricant is necessary in a tablet formulationto prevent the tablet and punches from sticking in the die. Thelubricant is chosen from such slippery solids as talc, magnesium andcalcium stearate, stearic acid and hydrogenated vegetable oils.

The preferred coating weights for particular coating materials may bereadily determined by those skilled in the art by evaluating individualrelease profiles for tablets, beads and granules prepared with differentquantities of various coating materials. It is the combination ofmaterials, method and form of application that produce the desiredrelease characteristics, which one can determine only from the clinicalstudies.

Methods of Treating Spinobulbar Muscular Atrophy

In various aspects, methods are provided for treating spinobulbarmuscular atrophy in a subject in need thereof. It is to be understoodthat, as spinobulbar muscular atrophy is a genetic disorder, treatingcan include impeding the progress of spinobulbar muscular atrophy,causing regression of the spinobulbar muscular atrophy, amelioration ofat least one symptom of spinobulbar muscular atrophy, even if theunderlying genetic disorder remains unaffected. Treating can includetreating the underlying pain associated with spinobulbar muscularatrophy even without alleviating additional symptoms.

The methods can include administering a therapeutically effective amountof a small molecule described herein or a derivative, prodrug, or saltthereof. The methods can include administering a pharmaceuticalformulation described herein. In some aspects, the administration is viaintravenous injection, intradermal injection, intramuscular injection,subcutaneous injection, infusion, or a combination thereof. In someaspects, the subject is a human subject.

Spinal and bulbar muscular atrophy is a gradually progressiveneuromuscular disorder in which degeneration of lower motor neuronsresults in muscle weakness, muscle atrophy, and fasciculation. Affectedindividuals often show gynecomastia, testicular atrophy, and reducedfertility. The therapeutically effective amount can effective to delayloss of body weight, a loss of mobility, and/or a loss of physicalstrength in the subject. The delay can include delaying the onset ascompared to a subject not receiving the treatment and/or decreasing therate of loss as compared to a subject not receiving the treatment. Thetherapeutically effective amount can be effective to prevent or delayneurogenic atrophy and/or to prevent a loss of spinal cord motor neuronsin the subject. The therapeutically effective amount can be effective torestore the frequency of type I myofibers to normal levels for a healthysubject. The therapeutically effective amount can be effective toreverse, delay the onset of, or delay the progression of testicularatrophy in the subject.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1: MEPB Yields Dose-Dependent Rescue from Loss of Body Weight,Rotarod Activity, and Grip Strength and Ameliorates Neuronal Loss,Neurogenic Atrophy, and Testicular Atrophy in SBMA

Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease causedby toxic gain-of-function of the androgen receptor (AR). Previousstudies have shown that coregulator binding through the activationfunction-2 (AF2) domain of AR may be a druggable target for selectivemodulation of toxic AR activity. Here we screened previously identifiedAF2 modulators for their ability to rescue toxicity in a Drosophilamodel of SBMA. We identified two compounds, tolfenamic acid (TA) and1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole(MEPB), as top candidates for rescuing lethality, locomotor function,and neuromuscular junction defects in SBMA flies. Pharmacokineticanalyses in mice showed good bioavailability of MEPB and poorbioavailability of TA in muscle, brain, and spinal cord. In apreclinical trial in a mouse model of SBMA, MEPB treatment yielded adose-dependent rescue from loss of body weight, rotarod activity, andgrip strength. In addition, MEPB ameliorated neuronal loss, neurogenicatrophy, and testicular atrophy, validating AF2 modulation as a potentandrogen-sparing strategy for SBMA therapy.

Materials and Methods

Study Design

The objective of this study was to determine whether modulation of theAR AF2 domain by small molecules that specifically bind to the BF3regulatory pocket would be a practicable therapy for improving the QOLof patients with SBMA. To this end, we screened several BF3-specificcompounds in Drosophila; performed a pilot preclinical study of the toptwo candidate compounds; performed an extensive, multidose preclinicalstudy of the lead compound; and determined the mechanism of action ofthese compounds in cell culture studies. Drosophila NMJ staining andanalysis was performed in a blinded manner, whereby treated larvae werecoded by an independent investigator. Treatment groups were not uncodeduntil analyses were completed. Both preclinical trials were performed ina blinded manner, whereby drugs (i.e., vehicle, TA, and/or MEPB) wereassigned a specific code by an independent investigator. Treatmentgroups remained blinded until data collection and analysis werecomplete. Drug dose and animal numbers were determined empirically forthe pilot preclinical study. The data acquired from this study were usedin a power analysis to estimate the number of animals required for thelarge preclinical study. Animal endpoints were reached when a mouseexhibited hindlimb paralysis/paresis or greater than 10% loss ofbodyweight, at which point the animal was humanely euthanized. Becausethe yield of AR121Q-positive male mice was generally low throughout ourstudies, randomization was not possible. Mice were entered intotreatment groups as they became available and were followedlongitudinally. Replicate number and statistical tests for each datasetare provided in the figure legends.

Reagents

For fly and cell culture experiments, TA (Sigma Aldrich), MEPB (Specs),bicalutamide (3B Scientific Corporation), ibuprofen (Sigma Aldrich), ordimethylcurcumin (Cayman Chemical) were resuspended in DMSO (SigmaAldrich), whereas DHT (Steraloids) was dissolved in ethanol, prior toaddition to fly food or culture media. All cell culture experiments wereperformed in media containing 10% charcoal-dextran stripped serum(Hyclone) to remove exogenous steroid hormones. For preclinical andpharmacokinetic mouse studies, TA and MEPB were dissolved in either a 10mg/mL (low dose) or 20 mg/mL (high dose) solution of corn oil containing1% DMSO at 37° C. for approximately 12 h, filtered (0.22 μm filter), andstored at 4° C. until injection.

Molecular Modeling

BF3-unbound AR LBD (pdb1T7R) in complex with an FXXLF motif was modeledusing PyMOL software. BF3 residues (1690, F691, P723, G724, N727, F826,E829, N833, E837, and R840) were highlighted green, charge clampresidues of the AF2 domain (K720, M734, M894, and E897) were highlightedred, and a synthetic FXXLF (FESLF) motif was highlighted blue. MEPBmodeling was performed in the same manner using pdb2YLO. TA modeling wasperformed in a similar manner using pdb2PIX, whereby flufenamic acid wasremoved and TA was added according the lowest predicted energyconformation using SwissDock software.

Fly Stocks and Phenotypic Characterization

UAS-AR52Q, UAS-AR52Q-K720A, and UAS-AR66Q-E897K fly stocks weregenerated as previously described⁸. Fly stocks were crossed to variousGAL4 driver lines (ELAV-GAL4 for pan-neuronal, OK371-GAL4 for motorneuron, GMR-GAL4 for eye) to induce expression of the AR transgene in atissue-specific manner in fly vials containing fly food with eithervehicle (1% ethanol+/−0.1% DMSO) or drugs. Upon the presence of progeny,parental flies were removed, and F1 flies were scored for phenotypicanalysis. Viability was determined as previously described⁴⁰. Briefly,the population frequency of adult F1 SBMA flies (ELAV>UAS-AR52Q orOK371>UAS-AR52Q) and control flies (GAL4-ELAV; CyO-GFP) were determinedby the presence or absence of the CyO phenotypic marker. At least threeindependent biological replicates (F0 crosses) were performed with newdrug/food preparations for each treatment group, and a total of at least50 flies were scored for each treatment group. To determine locomotoractivity of SBMA flies, adult F1 flies (OK371>UAS-AR52Q) reared on foodcontaining vehicle or drug were allowed to walk for 90 seconds in onewell of a 12-well tissue culture plate while video was recorded using aLeica M205C stereomicroscope and Leica DFC320 digital camera. Videoswere recorded for at least 15 flies per treatment group. Tracing of flymovement and analysis of displacement and velocity were performed usingImageJ software. Preparation and staining of larval neuromuscularjunctions and ventral ganglions were performed as previously described⁸.

Generation of AR121Q Transgenic Mice and Surgical Castration

Human AR cDNA containing 121 CAG/CAA alternating repeats was subclonedinto the pCAGGS vector. An 11.5 kb fragment containing the pCAGGSpromoter and AR cDNA was released by ApaL1 digestion, purified byQIAEXII (Qiagen), and injected into FVB pronuclei, which weretransferred into female FVB recipients. Founders were screened by DNAgenotyping of tail biopsies. Fluorescence in situ hybridization analysiswas performed on lung tissue to confirm transgene insertion. Out of 15founders, one line was viable, demonstrated germline transmission, andexpressed levels of hAR protein comparable with that of endogenous mouseAR. Therefore, the phenotype of this line was extensively characterized.Mice were maintained on a purebred FVB background for all studies.

Castration was performed according to USDA guidelines for aseptictechnique in animal survival surgery and oversight was provided byveterinary staff at St. Jude Children's Research Hospital. Briefly, micewere anesthetized by isoflurane exposure, and the surgical site wasprepared by shaving and disinfection. A 10-mm incision was made alongthe scrotal midline, and the testes were removed using forceps. Theconnection between the testes and vas deferens, as well the associatedblood vessels, were cauterized by hot forceps. Incisions were closedusing tissue glue, and preemptive analgesia (0.2 mg/kg meloxicam) wasadministered. Mice were bred and maintained in accordance with theguidelines set forth by the National Institutes of Health Guide for theCare and Use of Laboratory Animals, published by the U.S. Public HealthService. All experimental protocols were approved by the InstitutionalAnimal Care and Use Committee at St. Jude Children's Research Hospital.

Preclinical Trial Design in AR121Q Mice

To determine the efficacy of TA and/or MEPB in a mouse model of SBMA, wefirst performed a pilot study. Five mice were assigned to each drugtreatment group: 50 mg/kg TA, 50 mg/kg MEPB, or vehicle (1% DMSO in cornoil). Drug identities were coded to ensure investigator blinding, anddrugs were administered three times per week (Monday, Wednesday, andFriday) by intraperitoneal injection from 3 to 8 weeks of age. At 8weeks, all mice were sacrificed and tissues were collected forpathologic analysis. To fully characterize the efficacy of MEPB in SBMAmice, a large-scale, multi-dose preclinical trial was performed. A poweranalysis (Table 1) was performed based on the pilot study data todetermine the adequate number of mice required for 80% power. At least10 mice were assigned to each drug treatment group: 50 mg/kg MEPB, 100mg/kg MEPB, and vehicle (1% DMSO in corn oil). Drug identities werecoded to ensure investigator blinding, and drugs were administered threetimes per week (Monday, Wednesday, and Friday) by intraperitonealinjections from 4 weeks until 30 weeks of age.

TABLE 1 Statistical variables used to determine sample size Type IMinimum error Detection number of mice/ Assay rate Power differencegroup required Body weight 0.05 0.8 4 g 4 Rotarod 0.05 0.8 50 s 5 Gripstrength 0.05 0.8 20 g 10 Survival 0.05 0.8 47% 10

AR121Q Mouse Phenotypic Characterization

Body weight, rotarod activity, and grip strength data were collectedweekly. Footprint analysis and clasping phenotype were assessed at 7 and8 weeks of age. Body weight was measured using a standard laboratoryscale (Fisher Scientific). Rotarod activity analysis was performed on anaccelerating rotarod apparatus (IITC Life Science) using a two-day,weekly protocol. Mice were trained on the first day with one session setat 4 rpm for 5 min. The following day, mice were placed on theapparatus, rotation speed was set to accelerate from 4-40 rpm at a rateof 0.1 rpm/s, and the latency to fall was recorded for four separatetrials per mouse. Mice were given a 15-min rest period between eachtrial. Grip strength was measured using a grip strength meter (Bioseb).Grip strength was measured as grams of force in six repeatedmeasurements for forepaws and hindpaws of each animal. To performgait/footprint analysis, the forepaws and hindpaws of each animal weredipped in red and blue, respectively, water-soluble, non-toxic paint.The animal was then placed in a 70-cm long tunnel lined on the bottomwith Whatman filter paper, the entrance was sealed, and the animal wasallowed to walk through one time. Footprints were scanned and analyzedwith Image J for stride length and forepaw/hindpaw overlap⁴¹. Todetermine clasping phenotype, mice were recorded using an Apple Podcamera (iOS version 6.1.6) for approximately 60 seconds and still frameswere extracted using ImageJ.

AR121Q Mouse Pathologic Assessment

A separate cohort of five mice per treatment group was generated forpathologic and biochemical analysis. Mice were anesthetized byisoflurane inhalation and transcardially perfused with either PBS forfrozen tissue samples or 10% formalin for fixed tissue samples. Selectedtissues (brain, spinal cord, gastrocnemius/soleus muscle, testes, andliver) were dissected and either snap-frozen in liquid nitrogen orprocessed for paraffin embedding/sectioning. Tissue sections werestained with hematoxylin and eosin, toluidine blue, or Gomori trichrometo ascertain overall morphology and pathologic changes. To determine ARstaining and colocalization patterns, tissue sections were stained withanti-AR (N20, Santa Cruz), anti-polyQ (5TF1-1C2, EMD Millipore), oranti-ubiquitin (Cell Signaling Technology) antibodies. To quantify typeI and type II myofibers, hindlimb tissue sections were stained withmyosin heavy chain-slow twitch and myosin heavy chain-fast twitchantibodies (Leica Biosystems), respectively. For the purposes ofcounting ChAT-positive neurons, three stepped sections separated by 50μm were analyzed for the cervical, thoracic, and lumbosacral areas ofspinal cords. The slides contained three to six tissue sections at eachlevel and were labeled with an anti-ChAT antibody (EMD Millipore). Allslides were digitally scanned at scalable magnifications up to 20×(objective lens) using an Aperio XT Slide Scanner (Leica Biosystems).Whole slide images were imported into the Halo software program (IndicaLabs) and positive staining was quantified by an area quantificationalgorithm. For spinal cord motor neuron quantification, static imageswere made at a 2× magnification with ImageScope (Leica Biosystems) andthe number of ChAT-positive neurons located within the anterior hornwere counted with FIJI software⁴².

Pharmacokinetics

MEPB and TA concentrations in plasma, liver, muscle, testes, brain, andspinal cord of male FVB/NJ mice were measured at multiple time points (5min to 48 h) after a single intraperitoneal injection of either 100mg/kg body weight MEPB or 50 mg/kg body weight TA. MEPB or TA and theinternal standard (IS) SLV320 (Tocris Biosciences) were extracted fromplasma and tissue homogenate samples with methyl tert-butyl ether. A25-μL aliquot of sample was mixed with 10 μL IS (100 ng/mL). To this,600 μL methyl tert-butyl ether was added and vortexed for 5 min. Aftercentrifugation at 10,000 rpm for 5 min, the organic layer was thentransferred to a glass vial and dried in a CentriVap Console (Labconco)at 35° C. for 25 min. The dried extracts were then reconstituted with400 μL methanol, and an aliquot of 3 μL was injected onto thechromatographic system. All chemicals were HPLC grade or higher and wereobtained from Fisher Scientific (Fair Lawn) unless otherwise specified.Quantitation of MEPB and TA was performed using an API 4000 massspectrometer (AB SCIEX) equipped with a Prominence Ultra-Fast LiquidChromatograph (UFLCXR) system (Shimadzu). Chromatographic separation wasachieved with a Phenomenex Luna C18 column (3 μm, 100 A° 50×2.00 mm) byusing mobile phases consisting of 0.1% formic acid in water (A) andacetonitrile (B). Mass spectrometric analysis was performed with theturbo ion spray in positive ionization mode. All MS data were acquiredusing Analyst 1.5.2 software and processed using MultiQuant 2.1.1software (SCIEX).

For all matrices (plasma, liver, muscle, testes, brain, and spinalcord), the lower limit of quantification (LLOQ) of MEPB was 2.5 ng/mLwith a calibration range of 2.5 ng/mL to 100 ng/mL. However, due todilution during homogenization, the effective LLOQ was 15 ng/mL forsolid tissues. The LLOQ of TA was 5 ng/mL for plasma and 60 ng/mL fortissues. Assays were found to be linear and reproducible with acorrelation coefficient (R)>0.99. The MEPB concentration-time (Ct) datafor liver, muscle, testes, brain, and spinal cord were grouped by mouseand analyzed using a two-stage, semiphysiologic, nonlinear mixed effectsapproach with maximum likelihood expectation maximization in ADAPT 5.The pooled arithmetic mean Ct data were subjected to noncompartmentalanalysis by tissue using WinNonlin 6.4 (Pharsight, a Cetara Company).All PK parameters were calculated using standard formulae⁴³. Parametersestimated for all matrices included observed maximum concentration(Cmax), time of Cmax (Tmax), concentration at the last observed timepoint (Clast), time of Clast (Tlast), and area under the Ct curve (AUC).When applicable, the terminal phase was defined as the three time pointsat the end of the Ct profile, excluding Cmax, and the elimination rateconstant (Kel) was estimated using an unweighted log-linear regressionof the terminal phase. The terminal elimination half-life (T½) wasestimated as 0.693/Kel, and the AUC from time 0 to infinity (AUCinf) wasestimated as the AUC to the last time point (AUClast)+Clast/Ke. Apparentclearance (CL/F=Dose/AUCinf), and apparent terminal volume ofdistribution (Vz/F) were also calculated.

Immunoblotting and Immunofluorescence

Lysates from mouse tissues were prepared by grinding snap-frozen tissueson dry ice with a pestle until pulverized into a fine powder,resuspending in ice-cold RIPA (1% Triton-X, 0.1% sodium deoxycholate,and 0.1% SDS in Tris/NaCl) buffer, briefly sonicating, and centrifugingat 14,000 g for 20 min. Drosophila lysates were prepared as previouslydescribed⁸. Cell lysates were prepared by scraping transfected(mycoplasma-free) HEK293T cells (ATCC) or MN1 (first described by Brookset al. (1997)⁴⁴, gift from Kurt Fischbeck) cells into room temperaturePBS, centrifuging at 400 g for 5 min, and resuspending pellets inice-cold RIPA buffer, briefly sonicating, and centrifuging at 14,000 gfor 20 min. Supernatants were then collected, and protein levels weremeasured and adjusted using Bradford analysis. Proteins were separatedon 4% to 20% or 8% Tris-glycine gels and transferred overnight at 4° C.onto PVDF membranes. Membranes were stained with REVERT total proteinstain (Li-Cor) and immunoblotted using anti-AR (H280 or N20 [SantaCruz], D6F11 [Cell Signaling Technologies], or EPR1535(2) [Abcam]),anti-tubulin (Sigma Aldrich), anti-GAPDH (Cell Signaling Technologies),or anti-β-actin (Santa Cruz) antibodies. Filter trap assay foraggregation was performed by resuspending cell lysates in a 10% SDSbuffer to achieve a final 2% SDS concentration, heating for 5 min at 95°C., and applying to a 0.22 μm cellulose acetate membrane (GE HealthcareLife Sciences) with a vacuum dot blot apparatus (Schleicher andSchuell). Membranes were washed three times with 0.1% SDS and blottedwith anti-FLAG (M2, Sigma Aldrich). For immunofluorescence experimentsin Drosophila, UAS-AR flies were crossed to OK371-GAL4 flies at 25° C.on food containing either 1 mM DHT (Steraloids) or 1% ethanol togetherwith indicated drug. Third instar larvae were heat killed, dissected inPBS, and fixed with 4% PFA for 20 minutes. Primary antibody staining wasperformed at 4° C. overnight and secondary antibody staining wasperformed at room temperature for 4 hours. After staining, pelts weremounted in Fluoromount-G (SouthernBiotech). For immunofluorescenceexperiments in mice, deparaffinized and rehydrated cross-sections ofspinal cord and hindlimb muscle were permeabilized in 2% Triton-X 100,treated with TrueBlack autofluorescence quencher (Biotium), and blockedin PBS with 4% BSA and 2% NGS. Sections were incubated overnight at 4°C. with anti-NCAM (Proteintech), anti-PSA-NCAM (EMD Millipore),anti-ubiquitin (Abcam), or anti-polyQ (EMD Millipore) antibodies,followed by incubation for 2 h at room temperature with the appropriateAlexa Fluor-conjugated secondary antibodies (Thermo Fisher Scientific).Sections were mounted with ProLong Gold Antifade Mountant with DAPI(Thermo Fisher Scientific) and allowed to dry for at least 24 h at roomtemperature before imaging on a Leica TCS SP8 STED 3× confocalmicroscope (Leica Biosystems for ubiquitin-polyQ colocalization or LeicaDM18 Widefield microscope for NCAM) with 40× objective and LASX software(Leica).

NCAM Imaging and Analysis

One image was acquired in a region of three to four slices where brightpunctate NCAM and PSA-NCAM could be seen within the muscle fibers. If nopunctate regions greater than that of autofluorescence intensity ineither channel could be seen, a representative field without punctatesignal was taken for that muscle slice. Imaging and analysis wasperformed for two mice from each of the following conditions: untreatedNTG, untreated SBMA, NTG treated with 50 mg/kg MEPB, and NTG treatedwith 100 mg/kg MEPB. Three mice each were analyzed from each of thefollowing conditions: SBMA treated with 50 mg/kg MEPB and SBMA treatedwith 100 mg/kg MEPB. The same images were subjected to colocalizationanalysis with LASX software. For each image, the background out-of-focuslight was subtracted out and the remaining white detail was enhanced tomake the punctate regions more easily recognizable by the program.Following processing, an intensity-based mask was created for eachchannel, recognizing the regions where the signal was above that oftissue autofluorescence. These masks were then dilated and smoothed tocombine nearby punctate regions. Using a binary “AND” operand, thenumber of overlapping regions larger than 5 pixels that had signal inboth masks was calculated. Two-way ANOVA with Dunnett multiplecomparisons test was performed using GraphPad Prism version 6. P<0.05was considered significant.

Luciferase Reporter Assays

To determine AR-dependent transcriptional activity, HEK293T cells weretransiently transfected in culture media containing 10% charcoal-dextranstripped serum with ARE-firefly luciferase and CMV-Renilla luciferase(Cignal ARE Reporter Assay Kit, Qiagen), in addition to eitherFLAG-AR24Q or FLAG-AR65Q (gift from Maria Pennuto). Following 24 h oftransfection, cells were washed and treated with vehicle, bicalutamide,TA, or MEPB for 24 h. Firefly and Renilla luciferase substrates(Dual-Luciferase Reporter Assay, Promega) were added, and luciferaseactivity was measured using a microplate spectrophotometer (BioTek).Mammalian two-hybrid assays were performed by transiently transfectingHEK293T cells with pG5Luc firefly luciferase reporter (CheckmateMammalian Two-Hybrid kit, Promega), CMV-Renilla luciferase (Cignal AREReporter Assay Kit, Qiagen), and GAL4 DBD-AR LBD (gift from ElizabethWilson), in addition to either VP16 empty vector, VP16-NCoR, orVP16-SMRT (gifts from Vivian Bardwell) for 24 h. Cells were then washedand treated with vehicle, TA, or MEPB for 24 h. Renilla and fireflyluciferase were quantified using Dual-Luciferase Reporter Assay(Promega) and a microplate spectrophotometer (BioTek).

Droplet Digital PCR

The QX200 droplet digital PCR (ddPCR) system (Bio-Rad) was used tomeasure gene expression levels in 20 μl emulsion PCR reactions thatcontain 20,000 droplets. Total RNAs were firstly treated with DNase(Thermo Fisher Scientific, AM1907) to remove genomic DNA, and 5 ng oftreated RNA was used in each assay. ddPCR assay consisted of thefollowing components: 1× one-step RT-ddPCR mix for probes (Bio-Rad,1864021), forward primer (900 nM), reverse primer (900 nM), probe (FAMor VIC, 250 nM), nuclease-free water, and 5 ng RNA. All primers andprobes were purchased from Thermo Fisher Scientific. Droplets weregenerated in the droplet generator (Bio-Rad) and PCR was performed in aC1000 Touch thermal cycler (Bio-Rad) according to the manufacturer'srecommendation. After PCR, read-out of positive versus negative dropletswas performed using the QX200 droplet reader (Bio-Rad) and calculated byQuantaSoft software version 1.7.4.0917 (Bio-Rad).

Statistical Analyses

Significant changes in Drosophila population frequencies (i.e.,viability) were determined by Chi square analysis. Survival of SBMA micewas determined by Kaplan-Meier estimation, and comparisons betweensurvival curves were made with the log-rank (i.e., Mantel-Cox) test. Allother data, except power analysis and QOL score, were analyzed byone-way or two-way ANOVA and either Tukey or Dunnett post-hoc analysis,where appropriate, with Prism software (version 6.0, GraphPad). Poweranalysis was performed to determine sample size requirements to achieve80% power using SAS software (SAS Institute). QOL scores were determinedby averaging the change from baseline measurements for each behavioralphenotype (body weight, grip strength, and rotarod activity). A score ofzero was applied at each time point for any animal that could notcomplete the task due to hindlimb paralysis or that had been euthanized.A mixed effect model was applied using SAS software to determinestatistical significance.

Results

AF2 Modulation Rescues Degeneration in SBMA Flies

We compiled a panel of small-molecule compounds that were previouslyidentified by in silico or in vitro screening to modulate coregulatorbinding to the AR AF2 domain by specifically binding to a proximalregulatory pocket termed the binding function-3 (BF3) domain¹⁷⁻²². Uponbinding of such compounds to the BF3 domain, a conformational shiftoccurs in the AF2 domain that modulates the ability of coregulatorsbearing FXXLF and LXXLL motifs to bind the AF2 domain (FIG. 1A). Todetermine whether such compounds may be advantageous for SBMA therapy,we tested their ability to rescue DHT-induced lethality in fliesexpressing polyQ-expanded AR in pan-neuronal tissues (ELAV>UAS-AR52Q) orspecifically in motor neurons (OK371>UAS-AR52Q) (FIGS. 7A-7F). TA andMEPB treatment significantly increased SBMA fly viability in adose-dependent manner (FIGS. 1B-1C), similar to the known AR antagonistbicalutamide, whereas an AR-unrelated compound, ibuprofen, did not(FIGS. 7A-7F). Consistent with these results, both TA and MEPBsignificantly restored locomotor function in flies expressingpolyQ-expanded AR in motor neurons (OK371>UAS-AR52Q), measured asincreased displacement and velocity of walking in adult flies (FIGS.1D-1F), to similar levels observed in flies expressing AR variants thatmodulate coregulator binding to the AF2 domain (OK371>UAS-AR52Q-K720Aand OK371>UAS-AR66Q-E897K). Furthermore, both TA and MEPB restoredDHT-dependent neuromuscular junction defects in SBMA larvae ofOK371>UAS-AR52Q flies by reducing the prevalence of satellite boutonsand preventing loss of neuromuscular junction branching (FIGS. 1G-1I).These results were corroborated in flies expressing polyQ-expanded AR inthe eye (GMR>UAS-AR52Q), in which DHT-dependent degeneration wasmitigated by TA and MEPB, similar to that of bicalutamide anddimethylcurcumin (FIG. 8A), without significantly reducing monomeric oraggregated forms of AR protein levels (FIGS. 8B-8E)

Transgenic Male Mice Carrying Full-Length Human AR with 121 CAG RepeatsRecapitulate SBMA Symptoms and Pathology

Previously reported mouse models of SBMA that exhibit disease-relevantphenotypes express the polyQ-AR transgene at levels several-to-many foldhigher than endogenous AR. For example, the most frequently used mousemodel of SBMA¹² expresses exogenous human AR at levels approximatelythree times higher than endogenous AR levels (FIG. 9C and FIG. 9D).Although these prior mouse models of SBMA have been a valuable resourceto the research community, we were concerned about the possibility thathigh levels of mutant AR expression might mask the therapeutic potentialof SARMs. Thus, to evaluate the efficacy of SARM therapy in a mammaliansystem, we developed an animal model that phenocopies thedisease-relevant features of SBMA by expressing physiologically relevantlevels of mutant AR. Founder SBMA mice were produced by pronuclearinjection of human AR cDNA containing 121 CAG/CAA alternating repeatsdriven by the pCAGGS (CMV-IE enhancer+chick β-Actin) promoter (FIG. 9Aand FIG. 9B). We identified one line that expressed the AR transgene inspinal cord and muscle at endogenous levels (FIG. 9C and FIG. 9D) andthis line was chosen for further characterization.

Male AR121Q mice demonstrated rapid androgen-dependent declines in bodyweight, rotarod activity, grip strength, and survival (FIGS. 2A-2D) whencompared with nontransgenic FVB/NJ (NTG) control littermates.Immunohistochemistry using anti-AR, anti-polyQ, and anti-ubiquitinrevealed the presence of aggregates in the brain, spinal cord, andskeletal muscle but not in the testis or liver of AR121Q mice (FIGS.2E-2F and FIGS. 9E-9G). These ubiquitin-positive nuclear inclusions alsocolocalized with polyQ in the spinal cord and skeletal muscle (FIG. 2Gand FIG. 9H). Muscle fiber type switching from glycolytic type II fibersto oxidative type I fibers has recently been characterized as apathologic hallmark of muscle atrophy in mice and patients with SBMA²³.Consistent with this characterization, AR121Q mice displayed a markedpresence of atrophied skeletal muscle fibers and switching of musclefibers from glycolytic type I to oxidative type I (FIG. 2H-2I).Furthermore, we observed fewer choline acetyltransferase (ChAT)-positivemotor neurons in the anterior horn of the thoracic spinal cord in AR121Qmice compared to NTG controls (FIG. 2J-2K). Immunofluorescence withantibodies against neuronal cell adhesion molecule (NCAM) and itspolysialic acid form (PSA-NCAM), a protein known to be upregulatedduring muscle reinnervation²⁴⁻²⁶, revealed marked sarcoplasmic stainingof muscle fibers in AR121Q mice but not in NTG controls (FIG. 2L),demonstrating ongoing denervation/reinnervation in AR121Q mice. AR121Qmice also exhibited markedly decreased hindlimb muscle mass andincreased angular fibers (data not shown). Although centralized nucleiwere present in some muscle fibers of AR121Q mice (FIG. 2F),quantification revealed that both AR121Q and NTG mice had fewer than 3%of fibers with centralized nuclei, with no statistically significantdifference between them (FIG. 9I). Together, these data suggest thatAR121Q mice exhibit primarily neurogenic rather than myogenic atrophyand weakness.

Finally, we also observed testicular atrophy in the SBMA mice, similarto that described for an AR113Q knock-in mouse model of SBMA (FIG.9J)²⁷. This feature is consistent with the partial androgeninsensitivity experienced by patients with SBMA, which manifests astesticular atrophy, gynecomastia, and reduction in secondary sexualcharacteristics, and can impair QOL. The partial androgen insensitivityexperienced by SBMA patients is not reversed and may be aggravated byconventional, non-selective androgen ablation.

AF2 Modulation Improves Neurodegenerative Outcomes in a Pilot Study inSBMA Mice

To ascertain the potential efficacy of AF2 modulation in the SBMA mice,we performed a preclinical pilot assessment of the effects of AF2modulation on SBMA-associated degeneration in a small cohort of mice.Male SBMA mice were injected intraperitoneally (50 mg/kg body weight,three times per week) with TA, MEPB, or vehicle (1% DMSO in corn oil)from 3 to 8 weeks of age. MEPB treatment significantly improved bodyweight, rotarod activity, and grip strength (FIGS. 3A-3C), despite nosignificant change in survival (FIG. 3D), and improved gait and hindlimbclasping (FIGS. 3E-3G). In addition, the pathologic appearance ofskeletal muscle and spinal cord degeneration was qualitatively recoveredby MEPB treatment (FIGS. 3H-31 ). TA treatment did not significantlyalter any measurements of SBMA-associated degeneration (FIGS. 3A-31 ),suggesting that MEPB provides superior therapeutic potential in SBMAmice. Indeed, pharmacokinetic analysis of TA and MEPB in NTG micerevealed marked penetration and duration of MEPB but not TA in muscle,spinal cord, and testes (FIG. 10A and FIG. 10B, Table 2), indicatingsufficient bioavailability of MEPB in SBMA-affected tissues, but poorbioavailability of TA in SBMA mice. For this reason, MEPB was selectedfor subsequent proof of concept testing of the therapeutic potential ofAF2 modulation in SBMA mice.

TABLE 2 Maximum concentration (C_(max)), time of Cmax (T_(max)),concentration at the last observed time point (C_(last)), and time ofClast (T_(last)) of TA in plasma, liver, muscle, testes, brain, andspinal cord following a single intraperitoneal injection of 50 mg/kgbody weight TA. Estimate Variable Parameter Units Brain Liver MusclePlasma Spinal cord Testis TA conc. Cmax ng/mL 194 67600 1250 13400 5543030 Cmax μM 0.7 259.6 4.8 222.7 2.1 11.6 Tmax hr 1 1 1 1 1 1 Clastng/mL 66.7 508 119 19.8 84.6 150 Tlast hr 4 24 8 48 4 8

MEPB Demonstrates Efficacy in a Large-Scale Blinded Preclinical Trial inSBMA Mice

To further evaluate the therapeutic capacity of MEPB, we next performeda large-scale, blinded, multi-dose preclinical trial of MEPB treatmentin male SBMA mice. Using quantitative measures of SBMA-associateddegeneration (body weight, rotarod activity, grip strength, andsurvival) from the previous pilot study, we performed a statisticalpower analysis to establish cohort numbers required for a full-scaletrial (Table 1). On the basis of this analysis, we assigned at least 10male NTG or SBMA mice per treatment group. Mice were injectedintra-peritoneally with low-dose (50 mg/kg) MEPB, high-dose (100 mg/kg)MEPB, or vehicle (1% DMSO in corn oil) three times per week starting at4 weeks of age until completion of the trial when mice were 30 weeks ofage. Low-dose and especially high-dose MEPB treatment significantlyaugmented body weight, rotarod activity, and grip strength (FIGS. 4A-4K)and also qualitatively improved gait and hindlimb clasping (FIG. 11A andFIG. 11B). Consistent with improvement in these behavioral parameters,both low-dose and high-dose MEPB treatment significantly reduced thepresence of ubiquitin-positive nuclear inclusions in the spinal cord(FIGS. 5A-5B), as well as degenerating myofibers in skeletal muscle(FIGS. 5C-5D). Importantly, both low-dose and high-dose MEPB treatmentsignificantly reduced colocalization of NCAM/PSA-NCAM staining in SBMAmice as compared with vehicle-treated SBMA mice, decreasing these levelssimilar to those in NTG controls, consistent with amelioration ofneurogenic atrophy (FIGS. 5E-5F). Indeed, high-dose MEPB treatment wasfound to prevent the loss of spinal cord motor neurons (FIG. 11C andFIG. 11D). Moreover, both low-dose and high-dose MEPB treatment restoredthe frequency of type I myofibers to levels observed in NTG controlmice, whereas type II myofiber frequency remained unaffected (FIG. 11Eand FIG. 11F), further demonstrating the efficacy potential of AF2modulation for attenuation of SBMA-associated muscle degeneration.

Finally, testicular atrophy in the SBMA mice was reversed with bothlow-dose and high-dose MEPB treatment (FIG. 5G), which is not onlyconsistent with overall improvement of the SBMA phenotype, but alsounderscores the selective nature of AR modulation by MEPB.

Neither low-dose nor high-dose MEPB treatment significantly alteredsurvival of SBMA mice, although a dose-dependent trend in increasedsurvival was present (FIG. 4J). It should be noted that measurement ofmouse “survival” in this trial were confounded by protocol guidelinesdesigned to minimize morbidity in experimental animals. Mice werechecked daily by our veterinary staff and recommendations to euthanizeindividual animals were made based on loss of 10% body weight orsubjective signs of hindlimb weakness that might be severe enough tolimit an animal's ability to reach the feeder. However, when otherphenotypic measurements (viz., body weight, rotarod activity, and gripstrength) were compiled along with survival data to generate a QOLscore, high-dose MEPB treatment significantly improved the QOL of SBMAmice (FIG. 4K). Furthermore, MEPB treatment (low-dose or high-dose) didnot significantly change blood chemistry of either NTG or SBMA mice(FIGS. 11G-11I), and MEPB treatment (low-dose or high-dose) had nomeasurable effect on any assays of neuromuscular function in NTG controlmice, suggesting minimal MEPB-induced toxicity (FIGS. 4A-4K and FIG.11B). Together, these findings demonstrate that AF2 modulation by MEPBin SBMA mice improves multiple primary outcomes associated with reducedQOL in patients with SBMA, such as attenuated muscle strength,diminished coordination, and loss of body mass, without apparent adverseeffects.

AF2 Modulators do not Affect Normal AR Signaling Function, but PromoteCorepressor Binding

To assess the mechanism by which AF2 modulation attenuatesSBMA-associated phenotypes, we performed analyses of polyQ-expanded ARactivity in response to TA and MEPB (FIGS. 6A-6E and FIGS. 12A-12F).Neither TA nor MEPB treatment reduced steady state levels ofpolyQ-expanded AR protein in stably transfected motor neuron (MN1) cellsor transiently transfected HEK293T cells (FIGS. 12A-12E), indicatingthat reduced SBMA-associated toxicity as a result of TA and MEPBtreatment is not simply due to enhanced degradation of the AR. Inaddition, the presence of high-molecular weight, multimeric AR complexesand aggregates were unchanged in response to TA and MEPB treatment inHEK293T cells (FIG. 12E and FIG. 12F). Consistent with theseobservations, MEPB treatment did not statistically significantly changethe expression level of endogenous mouse AR or transgenic human AR inthe muscle and spinal cords of AR121Q mice (FIGS. 13A-13D). Similarly,neither MEPB nor TA treatment significantly changed the expression oftransgenic AR in Drosophila (FIG. 8D and FIG. 8E), suggesting that TA-and MEPB-mediated attenuation of toxicity occurs independently of ARaggregation. Moreover, neither MEPB nor TA treatment significantlyaltered DHT-dependent nuclear translocation of polyQ-expanded AR inHEK293T cells and Drosophila (FIGS. 6A-6B). A dual luciferase reporterassay demonstrated that the transactivational capacity of polyQ-expandedAR was not significantly altered by TA or MEPB treatment, although atrend of reduced transcriptional activity was present in cells treatedwith high concentrations (100 μM) of either TA or MEPB (FIG. 6C).Finally, we selected eight AR-responsive genes expressed in motorneurons (Igfbp5, Mt2, Sgk1, Trib1, Camkk2, Tsc22d3, Plk3r3, and AR) andassessed the impact of MEPB on response to ligand-dependent changes intranscription in MN1 cells that stably express human AR (MN1-AR24Qcells). We confirmed ligand-dependent changes in transcription for sevenof the eight target genes but found no impact of MEPB (FIG. 6D). Theseresults indicate that although the AR signaling pathway remains intact,AF2 modulation by SARMs selectively alters AR activity, reducing thetoxicity associated with polyQ-expanded AR but leaving other aspects ofAR responsiveness intact. Indeed, the reversal of testicular atrophy inthe SBMA mice upon treatment with MEPB is consistent with thisconclusion.

Because the AR has been well characterized to repress as well asactivate transcription of its target genes and these repressor/activatoractivities are dependent on binding of a specific complement ofcoactivators and corepressors to the AF2 domain¹⁰, we hypothesized thatSARM treatment may specifically promote recruitment of corepressors tothe AF2 domain rather than block coactivator binding. This wouldpotentially lead to repression of selected target genes drivingSBMA-associated toxicity, while allowing transcriptional activation ofother target genes. To determine whether association between the AR LBDand steroid receptor corepressors is augmented in response to AF2modulation by TA or MEPB, we performed a mammalian two-hybrid assay,whereby increased association between the AR LBD and a given coregulatordrives luciferase reporter activity. Consistent with our hypothesis,binding between the AR LBD and nuclear receptor corepressor-1 (NCoR) wasspecifically increased by TA and MEPB treatment, whereas binding ofsilencing mediator for retinoic acid and thyroid hormone receptors(SMRT) was unchanged (FIG. 6E), suggesting that modulation of the AF2domain by TA and MEPB occurs by a selective and precisely controlledmechanism.

DISCUSSION

Recent advances in prostate cancer research have led to the discovery ofmany SARM compounds that specifically bind to and modulate coregulatorbinding to the AR AF2 domain¹⁷⁻²². Interestingly, a reciprocalrelationship between prostate cancer and SBMA has been suggested to bemediated by polyQ length, as prostate cancer risk appears to be elevatedin individuals whose AR gene contains fewer than ten CAG repeats²⁸.Increased transactivational activity of the AR is also inverselycorrelated with CAG repeat length^(29,30), and polyQ expansion has beensuggested to augment intrinsic disorder within the N-terminal domain,reducing coactivator binding to the AR LBD through an unknownmechanism^(31,32). To avoid the proliferative effects of increased ARtransactivation, compounds that reduce coactivator binding or recruitcorepressor binding to the AF2 domain have been sought for prostatecancer therapy. The seminal discovery of the BF3 regulatory pocket as anallosteric regulator of the AF2 domain by Estebanez-Perpina et al.(2007) was an unexpected revelation in the search for such drugs¹⁹. Thedevelopment of several compounds that specifically target the BF3 pockethave since been reported²⁰⁻²².

A recent report by Jehle et al. (2014) demonstrated the importance ofthe BF3 domain in prostate cancer and the role that BF3 modulation mayplay in therapeutic treatment³³. Specifically, ectopic expression of theco-chaperone Bag-1L in the prostate secretory epithelium may beassociated with tumorigenesis by stimulating AR activation viainteraction between a duplicated GARRPR hexapeptide motif within Bag-1Land the AR BF3 binding pocket. Interestingly, the binding ofLXXLL-bearing coactivators to the AF2 domain was inhibited by Bag-1Lbinding to the BF3 pocket, further demonstrating the allostericregulation that BF3 imparts upon the AF2 domain. Moreover, BF3-Bag-1Lbinding was inhibited by the MEPB analog2-((2-(2,6-dimethylphenoxy)ethyl)thio)-1H-benzimidazole (compound 49 orCPD49)^(20,33), suggesting that modulation of the interaction betweenthe AR and Bag-1L by CPD49 may provide a promising approach to mitigatethe oncogenic program initiated by androgen signaling in prostatecancer.

We previously showed that coactivator binding to the AF2 domain isrequired for polyQ-expanded AR toxicity in Drosophila ⁸. Despite theapparent reciprocal relationship between prostate cancer and SBMA, wereasoned that targeting the BH3 interaction surface to modulatecoregulator binding to the AF2 domain may represent a common strategyfor treatment of both SBMA and prostate cancer. Therefore, we initiateda Drosophila-based screen to determine whether any previously describedBF3-binding compounds may be advantageous for the treatment of SBMA. Asexpected, several compounds either had no effect on, or evenexacerbated, SBMA toxicity (data not shown), suggesting that somecompounds may be more effective in the treatment of prostate cancerwhereas others may be more effective for SBMA. Nevertheless, twopromising compounds (TA and MEPB) emerged that amelioratedpolyQ-expanded AR toxicity in flies and became candidate therapeuticsfor a preclinical trial in a mammalian model of SBMA.

TA is a well-known and well-studied compound belonging to thenonsteroidal anti-inflammatory drug family of small molecules³⁴⁻³⁷.Although it has been approved for treatment of migraines in the UnitedKingdom by the National Health Service and is available as a generalanalgesic for humans and animals in several countries in Europe, LatinAmerica, and Asia, it has not been approved for any use in the UnitedStates. This compound was found to have poor bioavailability in mousebrain, spinal cord, and muscle and no significant effect in a pilottrial in AR121Q mice, and was subsequently not pursued in this study.

MEPB was first described by Lack et al. (2011) through virtual screeningfor BF3-binding compounds²¹. X-ray crystallography revealed MEPB tospecifically bind the BF3 pocket of the AR, where the benzimidazolemoiety of MEPB is oriented toward the interior of the BF3 pocket and isstabilized by strong hydrophobic interactions with Pro723, Phe673, andTyr834 and an arene-arene conjugation between a benzene ring of MEPB andPhe826. MEPB was shown unequivocally to bind to the BF3 domain and as aconsequence modulate AF2 binding to coregulators. MEPB was found to havegood bioavailability in mouse brain, spinal cord and muscle and, aftershowing a beneficial effect in a pilot trial in SBMA mice, was selectedfor further evaluation. In a blinded, multi-dose preclinical trial inmale SBMA mice, MEPB treatment was found to significantly augment bodyweight, reduce hindlimb clasping, and improve rotarod activity, gripstrength, gait, and QOL score. Consistent with improvement in thebehavioral phenotype, MEPB treatment rescued motor neuron loss andneurogenic atrophy. Finally, MEPB treatment was found to reversetesticular atrophy in the SBMA mice, a finding that underscores theselective activity of MEPB. Prior in vitro analyses found that MEPBbinding to BF3 enhances AF2 interaction with coregulators bearing anextended LXXLL motif (termed the “corepressor nuclear receptor box”),such as that found in the corepressor NCoR^(38,39). This observation isconsistent with our observation that NCoR was recruited to the AR LBD byMEPB, and suggests that MEPB may relieve polyQ-expanded AR-mediatedtoxicity by promoting the binding of corepressors to the AF2 domain.

Altogether, these results provide considerable evidence for the utilityof AR AF2 domain modulation by BF3-binding compounds as a paradigm forSBMA therapy. Subtle modulation of coregulator binding, and thus ARfunctional activity, rather than ablation of the entire androgensignaling pathway, may provide therapeutic relief of neurodegenerativesymptoms in patients with SBMA. Such a targeted approach may reduce orpotentially even reverse adverse effects of androgen insensitivity andimprove the QOL of patients with SBMA.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare set forth only for a clear understanding of the principles of thedisclosure. Many variations and modifications may be made to theabove-described embodiments of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure.

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The present disclosure will be better understood upon review of thefollowing features, which should not be confused with the claims.

Feature 1. A pharmaceutical formulation for treating spinobulbarmuscular atrophy in a subject in need thereof, the pharmaceuticalformulation comprising: a therapeutically effective amount of a smallmolecule, a derivative thereof, a prodrug thereof, or a salt thereof;wherein the small molecule has a structure according to the followingformula

wherein each occurrence of R¹, R², R³, and R⁴ is independently ahydrogen, a hydroxyl, a halogen, or a substituted or unsubstituted C₁-C₆alkyl or alkoxy; wherein each occurrence of X¹ and X² is independently Oor S; wherein each occurrence of A¹ is independently none or asubstituted or unsubstituted C₁-C₆ alkyl diradical; and wherein thetherapeutically effective amount is effective to ameliorate one or moresymptoms of spinobulbar muscular atrophy in the subject.

Feature 2. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein each occurrence of R¹ is a hydrogen.

Features 3. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein each occurrence of R² is a hydrogen.

Features 4. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein each occurrence of R³ is a hydrogen.

Features 5. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein R⁴ is a hydrogen, methyl, ethyl, isopropyl, ort-butyl.

Features 6. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein X¹ is S.

Features 7. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein each occurrence of X² is O.

Features 8. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein each occurrence of A¹ is an unsubstituted C₁-C₃alkyl diradical.

Features 9. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the small molecule is1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazolehaving a structure according to the following formula

Features 10. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the pharmaceutical formulation comprises thederivative of the small molecule; and wherein the derivative is selectedfrom the group consisting of ester and amide derivatives of the smallmolecule, pegylated derivatives of the small molecule, and N-oxides ofthe small molecule.

Features 11. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the pharmaceutical formulation comprises theprodrug of the small molecule; and wherein the prodrug is an amide,carbamate, imide, ester, anhydride, thioester, or thioanhydride of thesmall molecule.

Features 12. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the pharmaceutical formulation comprises thederivative of the small molecule, and wherein the derivative comprises asubstituent selected from the group consisting of a halogen, an azide,an alkyl, an aralkyl, an alkenyl, an alkynyl, a cycloalkyl, a hydroxyl,an alkoxyl, an amino, a nitro, a sulfhydryl, an imino, an amido, aphosphonate, a phosphinate, a carbonyl, a carboxyl, a silyl, an ether,an alkylthio, a sulfonyl, a sulfonamido, a ketone, an aldehyde, athioketone, an ester, a heterocyclyl, a —CN, an aryl, an aryloxy, aperhaloalkoxy, an aralkoxy, a heteroaryl, a heteroaryloxy, aheteroarylalkyl, a heteroaralkoxy, an azido, an alkylthio, an oxo, anacylalkyl, a carboxy esters, a carboxamido, an acyloxy, an aminoalkyl,an alkylaminoaryl, an alkylaryl, an alkylaminoalkyl, an alkoxyaryl, anarylamino, an aralkylamino, an alkylsulfonyl, a carboxamidoalkylaryl, acarboxamidoaryl, a hydroxyalkyl, a haloalkyl, an alkylaminoalkylcarboxy,an aminocarboxamidoalkyl, a cyano, an alkoxyalkyl, a perhaloalkyl, andan arylalkyloxyalkyl.

Features 13. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the pharmaceutical formulation comprises the saltof the small molecule, and either (i) the salt is a pharmaceuticallyacceptable acid addition salt comprising an anion selected from thegroup consisting of a sulfate, a citrate, matate, an acetate, anoxalate, a chloride, a bromide, an iodide, a nitrate, a sulfate, abisulfate, a phosphate, an acid phosphate, an isonicotinate, an acetate,a lactate, a salicylate, a tartrate, an oleate, a tannate, apantothenate, a bitartrate, an ascorbate, a succinate, a maleate, agentisinate, a fumarate, a gluconate, a glucaronate, a saccharate, aformate, a benzoate, a glutamate, a methanesulfonate, anethanesulfonate, a benzenesulfonate, a p-toluenesulfonate and a pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)); or (ii) the salt isa pharmaceutically acceptable base addition salt comprising a cationselected from the group consisting alkali metals and alkaline earthmetals, such as calcium, magnesium, sodium, lithium, zinc, potassium,and iron.

Features 14. A pharmaceutical formulation for treating spinobulbarmuscular atrophy in a subject in need thereof, the pharmaceuticalformulation comprising a therapeutically effective amount of a selectiveandrogen receptor modulator, wherein the therapeutically effectiveamount is effective to ameliorate one or more symptoms of spinobulbarmuscular atrophy in the subject.

Features 15. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the selective androgen receptor modulator altersa co-regulator binding to the activation function-2 (AF2) domain of theandrogen receptor.

Features 16. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the selective androgen receptor selectively bindsto the binding function-3 (BF3) domain of the androgen receptor.

Features 17. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the selective androgen receptor modulator isselected from the group consisting of17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one and derivatives thereof,testosterone and derivatives thereof, 4,5α-dihydrotestosterone andderivatives thereof,((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide)and a derivative thereof, and a combination thereof.

Features 18. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the formulation is a parenteral formulationselected from a solution, a suspension, an emulsion, and a solid formsuitable to prepare an injectable solution or suspension upon theaddition of a reconstitution medium.

Features 19. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the formulation is an enteral formulationselected from a tablet, a capsule, a solution, a suspension, a syrup,and a lozenge.

Features 20. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the effective amount is effective to prevent ordelay loss of body weight, a loss of mobility, and/or a loss of physicalstrength in the subject.

Features 21. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the effective amount is effective to prevent ordelay neurogenic atrophy and/or to prevent a loss of spinal cord motorneurons in the subject.

Features 22. The pharmaceutical formulation according to any one ofFeatures 1-23, wherein the effective amount is effective to restore thefrequency of type I myofibers to normal levels for a healthy subject.

Features 23. The pharmaceutical formulation according to any one ofFeatures 1-22, wherein the effective amount is effective to reversetesticular atrophy in the subject.

Feature 24. A method of treating spinobulbar muscular atrophy in asubject in need thereof, the method comprising administering atherapeutically effective amount of a small molecule, a derivativethereof, a prodrug thereof, or a salt thereof to the subject, whereinthe small molecule has a structure according to the following formula

wherein each occurrence of R¹, R², R³, and R⁴ is independently ahydrogen, a hydroxyl, a halogen, or a substituted or unsubstituted C₁-C₆alkyl or alkoxy; wherein each occurrence of X¹ and X² is independently Oor S; wherein each occurrence of A¹ is independently none or asubstituted or unsubstituted C₁-C₆ alkyl diradical; and wherein thetherapeutically effective amount is effective to ameliorate one or moresymptoms of spinobulbar muscular atrophy in the subject.

Feature 25. A method of treating spinobulbar muscular atrophy in asubject in need thereof, the method comprising administering atherapeutically effective amount of a selective androgen receptormodulator to the subject, wherein the therapeutically effective amountis effective to ameliorate one or more symptoms of spinobulbar muscularatrophy in the subject.

Feature 26. The method according to any one of Features 24-35, whereinthe selective androgen receptor modulator alters a co-regulator bindingto the activation function-2 (AF2) domain of the androgen receptor.

Feature 27. The method according to any one of Features 24-35, whereinthe selective androgen receptor selectively binds to the bindingfunction-3 (BF3) domain of the androgen receptor.

Feature 28. The method according to any one of Features 24-35, whereinthe selective androgen receptor modulator is selected from the groupconsisting of 17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one andderivatives thereof, testosterone and derivatives thereof,4,5α-dihydrotestosterone and derivatives thereof,((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide)and a derivative thereof, and a combination thereof.

Feature 29. The method according to any one of Features 24-35, whereinthe method comprises administering a pharmaceutical formulationaccording to any one of claims 1-23.

Feature 30. The method according to any one of Features 24-35, whereinthe effective amount is effective to prevent or delay loss of bodyweight, a loss of mobility, and/or a loss of physical strength in thesubject.

Feature 31. The method according to any one of Features 24-35, whereinthe effective amount is effective to prevent or delay neurogenic atrophyand/or to prevent a loss of spinal cord motor neurons in the subject.

Feature 32 The method according to any one of Features 24-35, whereinthe effective amount is effective to restore the frequency of type Imyofibers to normal levels for a healthy subject.

Feature 33. The method according to any one of Features 24-35, whereinthe effective amount is effective to reverse testicular atrophy in thesubject.

Feature 34. The method according to any one of Features 24-35, whereinthe subject is a human.

Feature 35. The method according to any one of Features 24-34, whereinthe method comprises intravenous injection, intradermal injection,intramuscular injection, subcutaneous injection, infusion, or acombination thereof.

1. A pharmaceutical formulation for treating spinobulbar muscularatrophy in a subject in need thereof, the pharmaceutical formulationcomprising: a therapeutically effective amount of a small moleculehaving a structure according to the following formula

or a derivative thereof, a prodrug thereof, or a salt thereof; whereineach occurrence of R¹, R², R³, and R⁴ is independently a hydrogen, ahydroxyl, a halogen, or a substituted or unsubstituted C₁-C₆ alkyl oralkoxy; wherein each occurrence of X¹ and X² is independently O or S;wherein each occurrence of A¹ is independently none or a substituted orunsubstituted C₁-C₆ alkyl diradical; and wherein the therapeuticallyeffective amount is effective to ameliorate one or more symptoms ofspinobulbar muscular atrophy in the subject.
 2. The pharmaceuticalformulation according to claim 1, wherein each occurrence of R¹ is ahydrogen.
 3. The pharmaceutical formulation according to claim 1,wherein each occurrence of R² is a hydrogen.
 4. The pharmaceuticalformulation according to claim 1, wherein each occurrence of R³ is ahydrogen.
 5. The pharmaceutical formulation according to claim 1,wherein R⁴ is a hydrogen, methyl, ethyl, isopropyl, or t-butyl.
 6. Thepharmaceutical formulation according to claim 5, wherein X¹ is S.
 7. Thepharmaceutical formulation according to claim 6, wherein each occurrenceof X² is O.
 8. The pharmaceutical formulation according to claim 1,wherein each occurrence of A¹ is an unsubstituted C₁-C₃ alkyl diradical.9. (canceled)
 10. The pharmaceutical formulation according to claim 1,wherein the pharmaceutical formulation comprises the derivative of thesmall molecule; and wherein the derivative is selected from the groupconsisting of ester and amide derivatives of the small molecule,pegylated derivatives of the small molecule, and N-oxides of the smallmolecule.
 11. The pharmaceutical formulation according to claim 1,wherein the pharmaceutical formulation comprises the prodrug of thesmall molecule; and wherein the prodrug is an amide, carbamate, imide,ester, anhydride, thioester, or thioanhydride of the small molecule.12-23. (canceled)
 24. A method of treating spinobulbar muscular atrophyin a subject in need thereof, the method comprising administering atherapeutically effective amount of a small molecule having a structureaccording to the following formula:

or a derivative thereof, a prodrug thereof, or a salt thereof; whereineach occurrence of R¹, R², R³, and R⁴ is independently a hydrogen, ahydroxyl, a halogen, or a substituted or unsubstituted C₁-C₆ alkyl oralkoxy; wherein each occurrence of X¹ and X² is independently O or S;wherein each occurrence of A¹ is independently none or a substituted orunsubstituted C₁-C₆ alkyl diradical; and wherein the therapeuticallyeffective amount is effective to ameliorate one or more symptoms ofspinobulbar muscular atrophy in the subject.
 25. A method of treatingspinobulbar muscular atrophy in a subject in need thereof, the methodcomprising administering a therapeutically effective amount of aselective androgen receptor modulator to the subject, wherein thetherapeutically effective amount is effective to ameliorate one or moresymptoms of spinobulbar muscular atrophy in the subject.
 26. The methodaccording to claim 25, wherein the selective androgen receptor modulatoralters a co-regulator binding to the activation function-2 (AF2) domainof the androgen receptor.
 27. The method according to claim 25, whereinthe selective androgen receptor selectively binds to the bindingfunction-3 (BF3) domain of the androgen receptor.
 28. The methodaccording to claim 25, wherein the selective androgen receptor modulatoris selected from the group consisting of17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one and derivatives thereof,testosterone and derivatives thereof, 4,5α-dihydrotestosterone andderivatives thereof,((2S)-3-(4-cyanophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methylpropanamide)and a derivative thereof, and a combination thereof.
 29. The methodaccording to claim 24, wherein the method comprises administering apharmaceutical formulation according to claim
 1. 30. The methodaccording to claim 24, wherein the effective amount is effective toprevent or delay loss of body weight, a loss of mobility, and/or a lossof physical strength in the subject.
 31. The method according to claim24, wherein the effective amount is effective to prevent or delayneurogenic atrophy and/or to prevent a loss of spinal cord motor neuronsin the subject.
 32. The method according to claim 24, wherein theeffective amount is effective to restore the frequency of type Imyofibers to normal levels for a healthy subject. 33-35. (canceled)